Hallucinogenic drug interactions at human brain 5-HT2 receptors: implications for treating LSD-induced hallucinogenesis

It has been shown that the hallucinogenic potencies of LSD, the phenylisopropylamines, such as DOB (4-bromo-2,5-dimethoxyphenylisopropylamine) and DOI (4-iodo-2,5-dimethoxyphenylisopropylamine), and the indoleaklylamines, such as DMT (dimethyltryptamine) and 5-OMe-DMT (5-methoxy-dimethyltryptamine), strongly correlate with their in vitro 5-HT₂ receptor binding affinities in rat cortical homogenates. In order to ascertain if this correlation applies to human 5-HT₂ receptors as well, we examined the affinities of 13 psychoactive compounds at ³H-ketanserin-labelled 5-HT₂ receptors in human cortical samples. Both radioligand binding and autoradiographical procedures were used. As in rat brain d-LSD was the most potent displacer of ³H-ketanserin specific binding with a K _i of 0.9 nM. The phenylisopropylamine DOI also displayed high affinity (K _i of 6 nM). Stereospecific interactions were found with DOB; (-_ DOB had a K _i of 17 nM while (+) DOB had a K _i of 55 nM. The behaviorally active compound DOM (4-methyl-2,5-phenylisopropylamine) had an affinity of 162 nM while its behaviorally less active congener iso-DOM had an affinity of 6299 nM. The indolealkylamines 5-OMe-DMT and DMT competed with moderate affinities (207 and 462 nM, respectively). In general, Hill coefficients were significantly less than unity which is consistent with an agonist interaction with 5-HT₂ receptors. MDMA, a substituted amphetamine analog was inactive with a K _i of greater than 10 M. A strong correlation was found for the hallucinogen affinities and human hallucinogenic potencies (r=0.97). Also, human and rat brain 5-HT₂ receptor affinities were strongly correlated (r=0.99). These results strongly support the hypothesis that the hallucinogenic effects of these drugs in humans are mediated in whole or in part via 5-HT₂ receptors. Furthermore, these studies imply that treatment with 5-HT₂ receptor antagonists may be effective in reversing the hallucinogenic effects caused by the ingestion, of LSD and LSD-like drugs.     

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.     

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD)

Rationale

After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however.

Objectives

Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model.

Method

Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection.

Results

Both DOI- and LSD-elicited head bobs required serotonin_2A (5-HT_2A) and dopamine₁ (D₁) receptor activation. Serotonin_2B/2C receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT_2A/2C receptor antagonist, ritanserin, bound frontocortical 5-HT_2A receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT_2A/2C receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D₁ receptor binding affinity whereas DOI had negligible binding affinity at this receptor.

Conclusion

Although DOI and LSD differed in their receptor binding properties, activation of 5-HT_2A and D₁ receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens.     

Evidence for involvement of 5-HT1C and 5-HT2 receptors in the food intake suppressant effects of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)

Administration of various doses of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) to rats produced dose-related decreases in 1-h food intake in the food-deprived paradigm. Pretreatment with spiperone (5-HT_1A/5-HT₂/D₂ antagonist), propranolol or CGP361A (-adrenoceptor antagonists that also have binding affinities for 5-HT_1A and 5-HT_1B sites) and MDL-72222 (5-HT₃ antagonist) did not attenuate DOI-induced suppression of food intake. In contrast, pretreatment with metergoline (5-HT₁/5-HT₂ antagonist) completely blocked whereas mesulergine, mianserin and ritanserin (5-HT_1C/5-HT₂ antagonists) partially blocked DOI's effect on food intake. On the other hand, pretreatment with MDL-72222 but not with m-chlorophenylpiperazine (m-CPP) significantly potentiated DOI-induced suppression of food intake. Furthermore, the food intake suppressant effects of various doses of DOI were found to be similar in the Fawn-Hooded (FH) rat strain as compared to the Wistar rat strain. These findings suggest that DOI-induced suppression of food intake is mediated by stimulation of both 5-HT_1C and 5-HT₂ receptors.     

Intrahippocampal LSD accelerates learning and desensitizes the 5-HT2A receptor in the rabbit, Romano et al.

Rationale  

Parenteral injections of d-lysergic acid diethylamide (LSD), a serotonin 5-HT_2A receptor agonist, enhance eyeblink conditioning. Another hallucinogen, (±)-1(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), was shown to elicit a 5-HT_2A-mediated behavior (head bobs) after injection into the hippocampus, a structure known to mediate trace eyeblink conditioning.     

The serotonin 2C receptor potently modulates the head-twitch response in mice induced by a phenethylamine hallucinogen

Rationale  

Hallucinogenic serotonin 2A (5-HT_2A) receptor partial agonists, such as (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT_2A receptor antagonists. In addition to 5-HT_2A receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT_2C receptors.     

Evidence that 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in rats is mediated by stimulation of 5-HT2A receptors

The effects of various 5-HT receptor subtype-selective antagonists were studied on phenylisopropylamine hallucinogen1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced hyperthermia in Wistar rats, in an attempt to characterize the 5-HT receptor subtype mediating DOI-induced hyperthermia. Intraperitoneal administration of DOI to rats produced hyperthermia with a peak effect at 60 min. Pretreatment with propranolol (-adrenoceptor antagonist that also has binding affinity for 5-HT_1A, 5-HT_1B and 5-HT_2C sites), MDL-72222 or ondansetron (5-HT₃ antagonists) did not attenuate DOI-induced hyperthermia. In contrast, pretreatment with metergoline (5-HT₁/5-HT₂ antagonist), ketanserin, LY53857, mesulergine, mianserin and ritanserin (5-HT_2C/5-HT_2A antagonists), as well as spiperone (5-HT_1A/5-HT_2A/D₂ antagonist), significantly attenuated DOI-induced hyperthermia. Furthermore, daily administration of DOI (2.5 mg/kg per day) for 17 days did not produce either tolerance to its hyperthermic effect or modifym-CPP-induced hyperthermia in rats. These findings suggest that DOI-induced hyperthermia in rats is mediated by stimulation of 5-HT_2A receptors.     

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.     

Age-related behavioural,neurochemical and radioligand binding changes in the central 5-HT system of Sprague-Dawley rats

Mature (3–4 months) and aged (18–19 months) Sprague-Dawley (SD) rats were treated with 5-HT receptor agonists and drug-induced behaviours monitored. The 5-HT_2/1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), induced wet dog shakes and back muscle contractions which were significantly increased in aged, compared to mature, rats, suggesting an age-related enhancement of 5-HT₂ receptor function. In contrast, the selective 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) induced forepaw treading, flat body posture, hypothermia and hyperactivity which were not significantly different in aged compared to mature rats. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus and frontal cortex were measured using high performance liquid chromatography with electrochemical detection. There were no age-related changes in hippocampal 5-HT or 5-HIAA. However both 5-HT and 5-HIAA were increased in the frontal cortex of aged SD rats. 8-OH-DPAT reduced 5-HIAA in both regions examined in mature rats, an effect which was attenuated in the aged rats, suggesting an age-related reduction in presynaptic 5-HT_1A receptor function. DOI did not induce any changes in 5-HT or 5-HIAA in either of the regions examined. Radioligand binding studies with [³H] ketanserin showed there to be no significant age-related changes in cortical 5-HT₂ receptor density or affinity. In the samples taken from mature rats GTP shifted the competition curve to DOI and reduced the proportion of high affinity agonist binding sites; this effect was not observed in the aged samples, suggesting that there may be age-related changes in G-protein-mediated receptor-effector coupling mechanisms.     

Novel regulation of 5-HT1C receptors: down-regulation induced both by 5-HT1C/2 receptor agonists and antagonists

The 5-hydroxytryptamine_1C (5-HT_1C) receptor shares many features with the 5-HT₂ receptor. To determine if the regulation of the sites is also similar we studied the effects of chronic treatment with drugs active at 5-HT_1C/2 receptors on [³H]mesulergine-labelled 5-HT_1C binding sites in spinal cord. The 5-HT receptor agonists 1-(3-chlorophenyl)piperazine (m-CPP) (-38%), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (-35%), quipazine (-27%) and m-trifluoromethylphenylpiperazine (TFMPP) (-27%) significantly down-regulated spinal 5-HT_1C sites with chronic injection compared to vehicle treatment. The 5-HT receptor antagonist methiothepin (-71%), mianserin (-24%), methysergide (-21%), and cyproheptadine (-27%) also induced down-regulation, and ritanserin and metergoline further reduced [³H]mesulergine specific binding to undetectable levels. There were no significant changes in K_d to implicate presence of residual drug except for mianserin, methiothepin, and TFMPP. Pindolol and spiperone had no significant effects. In acute dose-response studies, injection of a single dose of DOI did not result in a significant change in any receptor parameters. The capacity of a drug to lower B_max correlated significantly with its pK_d (r = 0.84, P < 0.0007). This drug regulation pattern for 5-HT_1C sites of down-regulation by both 5-HT_1C/2 receptor agonists and antagonists is similar to that for 5-HT₂ receptors and is consistent with the classification of 5-HT_1C and 5-HT₂ receptors in the same superfamily.     

Evidence thatm-chlorophenylpiperazine-induced hyperthermia in rats is mediated by stimulation of 5-HT2C receptors

Intraperitoneal administration ofm-chlorophenylpiperazine (m-CPP) to Wistar rats produced hyperthermia with a peak effect at 30 min. Pretreatment with low doses of metergoline (5-HT₁/5-HT₂ antagonist), mesulergine and mianserin (5-HT_2C/5-HT_2A antagonists) blockedm-CPP-induced hyperthermia. Pretreatment with propranolol (-adrenergic receptor antagonist that also has binding affinity for 5-HT_1A, 5-HT_1B and 5-HT_2B sites), yohimbine (₂-noradrenergic antagonist that also has binding affinity for 5-HT_2B sites), MDL-72222 or ondansetron (5-HT₃ antagonists) did not attenuatem-CPP-induced hyperthermia. Only high doses of ketanserin, LY-53857 and ritanserin (5-HT_2A/5-HT_2C antagonists) as well as spiperone (5-HT_1A/5-HT_2A/D₂ antagonist) attenuatedm-CPP-induced hyperthermia. Daily administration ofm-CPP produced complete tolerance to its hyperthermic effect by day 5. However, there was no cross-tolerance to 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, a 5-HT_2A agonist that also has high affinity for 5-HT_2C receptors)-induced hyperthermia.m-CPP-induced increases in temperature were found to be significantly less in the Fawn-Hooded (FH) rat strain as compared to the Wistar rat strain; in prior studies, FH rats have been found to be subsensitive to other 5-HT_2C-mediated pharmacologic responses. Altogether, these findings suggest thatm-CPP-induced hyperthermia in rats is mediated by selective stimulation of 5-HT_2C receptors.     

Mefloquine and psychotomimetics share neurotransmitter receptor and transporter interactions in vitro

Rationale

Mefloquine is used for the prevention and treatment of chloroquine-resistant malaria, but its use is associated with nightmares, hallucinations, and exacerbation of symptoms of post-traumatic stress disorder. We hypothesized that potential mechanisms of action for the adverse psychotropic effects of mefloquine resemble those of other known psychotomimetics.

Objectives

Using in vitro radioligand binding and functional assays, we examined the interaction of (+)- and (?)-mefloquine enantiomers, the non-psychotomimetic anti-malarial agent, chloroquine, and several hallucinogens and psychostimulants with recombinant human neurotransmitter receptors and transporters.

Results

Hallucinogens and mefloquine bound stereoselectively and with relatively high affinity (K _i?=?0.71–341 nM) to serotonin (5-HT) _2A but not 5-HT_1A or 5-HT_2C receptors. Mefloquine but not chloroquine was a partial 5-HT_2A agonist and a full 5-HT_2C agonist, stimulating inositol phosphate accumulation, with similar potency and efficacy as the hallucinogen dimethyltryptamine (DMT). 5-HT receptor antagonists blocked mefloquine’s effects. Mefloquine had low or no affinity for dopamine D₁, D₂, D₃, and D_4.4 receptors, or dopamine and norepinephrine transporters. However, mefloquine was a very low potency antagonist at the D₃ receptor and mefloquine but not chloroquine or hallucinogens blocked [³H]5-HT uptake by the 5-HT transporter.

Conclusions

Mefloquine, but not chloroquine, shares an in vitro receptor interaction profile with some hallucinogens and this neurochemistry may be relevant to the adverse neuropsychiatric effects associated with mefloquine use by a small percentage of patients. Additionally, evaluating interactions with this panel of receptors and transporters may be useful for characterizing effects of other psychotropic drugs and for avoiding psychotomimetic effects for new pharmacotherapies, including antimalarial quinolines.     

Indolealkylamine analogs share 5-HT2 binding characteristics with phenylalkylamine hallucinogens

Twenty-one indolealkylamines, some of which are known to be psychoactive in man, were examined for their binding interactions with rat brain cortical 5-HT2 receptors labeled with the antagonist radioligand [3H]ketanserin in order to develop structure-activity relationships for binding at these sites. Features investigated included aromatic, alpha-methyl and terminal amine substituents. 4-Methoxy and 5-methoxy substitution impart a higher affinity than 6- or 7-methoxy substitution; a 7-hydroxyl group essentially abolishes affinity whereas a 7-methyl or 7-bromo group enhances affinity. alpha-Methylation has little effect on affinity and, in the one case examined, the S(+) isomer of alpha-methyltryptamine was essentially equipotent with its racemate and twice as potent as its R(-) enantiomer. Terminal amine methylation results in a small but progressive decrease in affinity in the order: primary amine greater than dimethylamine greater than diethylamine. Similarities were noted between these structural requirements for binding and those of the phenalkylamines. Selected compounds (5-methoxytryptamine, N,N-dimethyltryptamine, 5-methoxy-N,N-diethyltryptamine and 5-methoxy-N,N-dimethyltryptamine) were further examined by two-site analysis of displacement studies for [3H]ketanserin specific binding. Hill coefficients were significantly less than unity and computer-assisted analysis indicated that a two-site model better fit the data than a one-site model. In displacement studies using the putative agonist radioligand [3H]DOB to label 5-HT2 receptors affinities were 10-100-fold higher than those using [3H]ketanserin. These results are also consistent with earlier findings using psychoactive phenalkylamines in competition studies for radiolabelled 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic inhibition of the mossy fibre—granule cell glutamate transmission in rat cerebellar slices

Summary The glutamatergic mossy fibre granule cell pathway has been investigated in rat cerebellar slices. Exposure to 35 mM KCI, a concentration of K⁺ known to elicit Ca²⁺-dependent releases of excitatory amino acids from cerebellar slices, raised cGMP levels. The cGMP response was decreased in a concentration-dependent manner by D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5) and by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicating the involvement of ionotropic glutamate receptors of both the N-methyl-D-aspartate (NMDA) and the non-NMDA type. The K⁺-evoked production of cGMP was potently inhibited (EC₅₀ = 1.21 nM) by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a selective 5-HT₂ receptor agonist. The effect of DOI (0.01 M) was antagonized by 0.03 M of the 5-HT₂ receptor antagonists ketanserin and methiothepin. At concentrations higher than 0.1 M, both antagonists increased on their own the cGMP response elicited by high-K⁺. This effect was insensitive to tetrodotoxin.It had been previously shown that rat mossy fibre endings release glutamate upon depolarization and that such release can be inhibited by activation of 5-HT₂ receptors sited on the mossy fibre endings. Altogether the available data suggest the following conclusions: (a) the glutamate/aspartate endogenously released in cerebellar slices during K⁺ depolarization increase cGMP synthesis through the activation of both NMDA and non-NMDA receptors; (b) a portion of the cGMP response can be prevented by 5-HT₂ receptor activation and may reflect the activity of the mossy fibre-granule cell pathway. Thus serotonin is likely to exert a potent inhibitory control of the excitatory mossy fibre input to the cerebellum by acting at receptors of the 5-HT₂ type. Correspondence to M. Raiteri at the above address     

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.     

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     

Promotion of cell growth by stimulation of cloned human 5-HT1D receptor sites in transfected C6-glial cells is highly sensitive to intrinsic activity at 5-HT1D receptors

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT_1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT_1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144–156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC₅₀: 25 nM) promoted cell growth of C6-glial/5-HT_1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC₅₀ in nM): zolmitriptan (0.41), 2-methyl-4-(5-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127,935; 0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-y)]ethylamine (MK-462; 3.0), and R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC₅₀-values correspond to the compounds binding affinities at the human 5-HT_1D receptor site and, with the exception of GR 127,935 and metergoline, also to the EC₅₀-values found by measuring over 5 min inhibition of forskolin (100 M)-stimulated cAMP formation. Prolonged exposure of GR 127,935 (3 h) and metergoline (30 min) to cells yielded EC₅₀ values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonists methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated CAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT_1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 M) but not by spiperone (1 M). Micromolar concentrations of the 5-HT_1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3, 2-b]pyril-5-one (CP 93,129) and of the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT_1D receptor site. These effects were sensitive to ketanserin (1 M) antagonism, but not to blockade by -adrenergic blockers and the 5-HT₂ receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT_1A, 5-HT_1B and 5-HT₂ receptors are not implicated in 5-HT-stimulated C6-glial/5-HT_1D cell growth. In conclusion, human 5-HT_1D receptors are involved in the growth of C6-glial/5-HT_1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT_1D receptors.     

Behavioral and neurochemical pharmacology of six psychoactive substituted phenethylamines: mouse locomotion,rat drug discrimination and in vitro receptor and transporter binding and function

Rationale

Psychoactive-substituted phenethylamines 2,5-dimethoxy-4-chlorophenethylamine (2C-C); 2,5-dimethoxy-4-methylphenethylamine (2C-D); 2,5-dimethoxy-4-ethylphenethylamine (2C-E); 2,5-dimethoxy-4-iodophenethylamine (2C-I); 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2); and 2,5-dimethoxy-4-chloroamphetamine (DOC) are used recreationally and may have deleterious side effects.

Objectives

This study compares the behavioral effects and the mechanisms of action of these substituted phenethylamines with those of hallucinogens and a stimulant.

Methods

The effects of these compounds on mouse locomotor activity and in rats trained to discriminate dimethyltryptamine, (?)-DOM, (+)-LSD, (±)-MDMA, and S(+)-methamphetamine were assessed. Binding and functional activity of the phenethylamines at 5-HT_1A, 5-HT_2A, 5-HT_2C receptors and monoamine transporters were assessed using cells heterologously expressing these proteins.

Results

The phenethylamines depressed mouse locomotor activity, although 2C-D and 2C-E stimulated activity at low doses. The phenethylamines except 2C-T-2 fully substituted for at least one hallucinogenic training compound, but none fully substituted for (+)-methamphetamine. At 5-HT_1A receptors, only 2C-T-2 and 2C-I were partial-to-full very low potency agonists. In 5-HT_2A arachidonic acid release assays, the phenethylamines were partial to full agonists except 2C-I which was an antagonist. All compounds were full agonists at 5-HT_2A and 5-HT_2C receptor inositol phosphate assays. Only 2C-I had moderate affinity for, and very low potency at, the serotonin transporter.

Conclusions

The discriminative stimulus effects of 2C-C, 2C-D, 2C-E, 2C-I, and DOC were similar to those of several hallucinogens, but not methamphetamine. Additionally, the substituted phenethylamines were full agonists at 5-HT_2A and 5-HT_2C receptors, but for 2C-T-2, this was not sufficient to produce hallucinogen-like discriminative stimulus effects. Additionally, the 5-HT_2A inositol phosphate pathway may be important in 2C-I's psychoactive properties.     

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     

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.