Distribution of 5-ht(1E) receptors in the mammalian brain and cerebral vasculature: an immunohistochemical and pharmacological study.

BACKGROUND AND PURPOSE: The 5-ht(1E) receptor is highly expressed in the human brain and its structure is conserved in humans, suggesting an important physiological role for 5-ht(1E) receptors. However, neither the function nor the distribution of this receptor has been characterized in the mammalian brain. EXPERIMENTAL APPROACH: Rats and mice lack the 5-ht(1E) receptor gene; thus, we used guinea pig brain tissue and immunofluorescent staining techniques to provide the first specific localization of 5-ht(1E) receptors in the mammalian brain. KEY RESULTS: High levels of 5-ht(1E) receptors are detected in olfactory bulb glomeruli as well as the molecular layer of the dentate gyrus (DG). In DG membranes, BRL54443, a 5-ht(1E) /5-HT(1F) agonist, selectively stimulated 5-ht(1E) receptors and potently inhibited forskolin-dependent cAMP production (IC50 = 14 nM). The staining pattern of 5-ht(1E) receptors in brain tissue suggests that this receptor is expressed predominantly in neurons rather than in glia. Additionally, 5-ht(1E) receptors were detected in the adventitial layer of cerebral arteries but not in the microvasculature, venous tissue or other brain arteries. CONCLUSIONS AND IMPLICATIONS: These observations should help to predict clinical effects of 5-ht(1E) -selective drugs. For example, the stimulation of 5-ht(1E) receptors and subsequent inhibition of adenylate cyclase activity in the DG suggests that 5-ht(1E) receptors may mediate regulation of hippocampal activity by 5-HT, making it a possible drug target for the treatment of neuropsychiatric disorders characterized by memory deficits (such as Alzheimer's disease) or as a target for the treatment of temporal lobe epilepsy.

Antagonist interaction with the human 5-HT(7) receptor mediates the rapid and potent inhibition of non-G-protein-stimulated adenylate cyclase activity: a novel GPCR effect.

BACKGROUND AND PURPOSE: The human 5-hydroxytryptamine(7) (h5-HT(7)) receptor is G(s) -coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT(7) receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other G(s) -coupled receptors. EXPERIMENTAL APPROACH: Recombinant cell lines expressing h5-HT(7) receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay. KEY RESULTS: In human embryonic kidney 293 cells stably expressing h5-HT(7) receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not G(i/o) -mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT(7) binding affinity (r(2) = 0.91), indicating that the antagonists acted through h5-HT(7) receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT(7) antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT(6), human ß(1), ß(2), and ß(3) adrenoceptors did not inhibit forskolin's activity. CONCLUSIONS AND IMPLICATIONS: The inhibition of adenylate cyclase, as measured by forskolin's activity, is an underlying property of antagonist interaction with h5-HT(7) receptors; however, this is not a common property of other G(s) -coupled receptors. This phenomenon may be involved in the roles played by h5-HT(7) receptors in human physiology. Development of h5-HT(7) antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance.

Behavioral characterization of 2-O-desmethyl and 5-O-desmethyl metabolites of the phenylethylamine hallucinogen DOM.

The present investigation was undertaken to test the hypothesis that known metabolites of the phenylethylamine hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) are pharmacologically active. This hypothesis was tested by evaluating the ability of racemic DOM metabolites 2-O-desmethyl DOM (2-DM-DOM) and 5-O-desmethyl DOM (5-DM-DOM) to substitute for the stimulus properties of (+)lysergic acid diethylamide (LSD). The data indicate that both metabolites are active in LSD-trained subjects and are significantly inhibited by the selective 5-HT(2A) receptor antagonist M100907. Full generalization of LSD to both 2-DM-DOM and 5-DM-DOM occurred, and 5-DM-DOM was slightly more potent than 2-DM-DOM. Similarly, 5-DM-DOM had a slightly higher affinity than 2-DM-DOM for both 5-HT(2A) and 5-HT(2C) receptors. Additionally, it was of interest to determine if the formation of active metabolite(s) resulted in a temporal delay associated with maximal stimulus effects of DOM. We postulated that if metabolite formation resulted in the aforementioned delay, direct administration of the metabolites might result in maximally stable stimulus effects at an earlier pretreatment time. This hypothesis was tested by evaluating (1) the time point at which DOM produces the greatest degree of LSD-appropriate responding, (2) the involvement of 5-HT(2A) receptor in the stimulus effects of DOM at various pretreatment times by administration of M100907 and (3) the ability of 2-DM-DOM and 5-DM-DOM to substitute for the stimulus properties of LSD using either 15- or 75-min pretreatment time. The data indicate that (a) the DOM stimulus produces the greatest degree of LSD-appropriate responding at the 75-min time point in comparison with earlier pretreatment times and (b) the stimulus effects of DOM are differentially antagonized by M100907 and this effect is a function of DOM pretreatment time prior to testing. Both 2-DM-DOM and 5-DM-DOM were found to be most active, at all doses tested, using a 75-min versus a 15-min pretreatment time. The present data do not permit unequivocal acceptance or rejection of the hypothesis that active metabolites of (-)-DOM provide a full explanation of the observed discrepancy between brain levels of (-)-DOM and maximal stimulus effects.

1-[2-methoxy-5-(3-phenylpropyl)]-2-aminopropane unexpectedly shows 5-HT(2A) serotonin receptor affinity and antagonist character.

Certain phenylethylamines, such as 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a), are high-affinity 5-HT(2) agonists. Previous structure-affinity studies have concluded that both the 2,5-dimethoxy substitution pattern and the nature of substituents at the 4-position are important determinants of high affinity. We recently demonstrated that replacement of the bromo group of DOB with a 3-(phenyl)propyl substituent results in retention of affinity and that, counter to established structure-affinity relationships, the 2,5-dimethoxy substitution pattern is no longer a requirement for the binding. The present investigation extends these findings by examining a series of analogues, 3, lacking a 5-methoxy group. It was additionally found that shifting the phenylalkyl substituent from the 4- to the 5-position (e.g., 4i) also results in retention of affinity. For example, 1-(2-methoxy-5-(3-phenylpropyl)-2-aminopropane (6; the alpha-methyl derivative of 4i) binds at 5-HT(2A) receptors with high affinity (K(i) = 13 nM) and possesses 5-HT(2A) antagonist character. Thus, not only is the 2,5-dimethoxy substitution pattern not a requirement for the binding of certain phenylethylamines at 5-HT(2A) receptors, the presence of a 4-position substituent (previously thought to serve as a modulator of affinity of DOB-like agents) is also not required. Striking differences in the 5-HT(2A) binding requirements of the present compounds as compared to DOB-like agents suggest multiple substituent-dependent modes of binding.

The binding of arylguanidines at 5-HT(3) serotonin receptors: a structure-affinity investigation.

The 5-HT(3) receptor binding affinities of nine pairs of aryl-substituted arylguanidines and arylbiguanides were examined and the results suggest the likelihood that both classes of agents utilize common receptor binding features. The effects of structural modification were also examined using CoMFA. 1-(3,4,5-Trichlorophenyl)guanidine (5-HT(3) K(i)=0.7 nM) was identified as a very high-affinity arylguanidine. The structures of the high-affinity arylguanidines are inconsistent with current 5-HT(3) pharmacophore models.

Behavioural sensitization to cocaine is dissociated from changes in striatal NMDA receptor levels.

Changes in glutamate transmission and alterations in glutamate receptor expression produced by the repeated administration of psychomotor stimulant drugs are considered an important neuroadaptation underlying the development and expression of behavioural and neurochemical sensitization to stimulant drugs. Two parallel experiments investigated the effects of repeated cocaine administration (five, once daily injections of 15 mg/kg, i.p.; 2 weeks withdrawal) on the expression of behavioural sensitization in response to a cocaine challenge (20 mg/kg, i.p.) and the changes in NMDA receptor binding in pooled tissue from the nucleus accumbens and the striatum. Compared with acute cocaine controls (n = 11), animals administered cocaine repeatedly displayed a sensitized stereotypic response to the cocaine challenge injection (n = 8). Despite this, no differences in either NMDA receptor density or affinity were observed between rats administered repeatedly with cocaine or saline, as indexed by [3H]MK-801 binding. The present findings call to question the rationale for NMDA receptor-based pharmacotherapies for the treatment of the enduring symptomatology of stimulant addiction.

Inverse agonist activity of atypical antipsychotic drugs at human 5-hydroxytryptamine2C receptors.

Clozapine is the prototype atypical antipsychotic drug, producing little or no extrapyramidal side effects, while improving negative symptoms of psychosis. Clozapine's high affinity for serotonin receptors has been hypothesized to confer the unique antipsychotic properties of this drug. Recently, we demonstrated that both typical and atypical antipsychotic drugs are inverse agonists at constitutively active 5-hydroxytryptamine2A (5-HT(2A)) receptors. To determine whether inverse agonist activity at 5-HT(2C) receptors plays a role in antipsychotic efficacy, typical and atypical antipsychotic drugs were tested for inhibition of basal inositol phosphate production in mammalian cells expressing rat or human 5-HT(2C) receptors. Atypical antipsychotic drugs (sertindole, clozapine, olanzapine, ziprasidone, risperidone, zotepine, tiospirone, fluperlapine, tenilapine) displayed potent inverse agonist activity at rat and human 5-HT(2C) receptors. Typical antipsychotic drugs (chlorpromazine, loxapine, thioridazine, prochlorperazine, perphenazine, mesoridazine, trifluperidol, fluphenazine, spiperone, haloperidol, pimozide, penfluridol, thiothixene) were devoid of inverse agonist activity, with the exception of loxapine. We review the evidence that loxapine has unique properties characteristic of both atypical and typical antipsychotic drugs. Several typical antipsychotic drugs (chlorpromazine, thioridazine, spiperone, thiothixene) displayed neutral antagonist activity by reversing clozapine inverse agonism. These data suggest that 5-HT(2C) inverse agonist activity is associated with atypical antipsychotic drugs with moderate to high affinity for 5-HT(2C) receptors, and imply that effects of atypical antipsychotic drugs on the 5-HT(2C) receptor may play a role in their unique clinical properties. These data also imply that dysfunction of brain 5-HT(2C) receptor systems may be one of the factors involved in the etiology of psychosis.

1-[4-(3-Phenylalkyl)phenyl]-2-aminopropanes as 5-HT(2A) partial agonists.

Phenylalkylamines such as 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) and its corresponding iodo derivative DOI (2) are commonly used 5-HT(2) serotonin agonists. Previous studies have established that the 2,5-dimethoxy substitution pattern found in these compounds is optimal for high affinity at 5-HT(2A) receptors and that substituents at the 4-position can modulate affinity over a wide range. We have previously shown, however, that when the 4-position is substituted with a 3-phenylpropyl substituent (i.e., 3), the compound binds with an affinity comparable to that of 1a but that it possesses 5-HT(2A) antagonist character. The present study examined the structure-affinity relationships of 3, and the results were very much unexpected. That is, the 2,5-dimethoxy substitution pattern of 3 is not required for high affinity. Either of the two methoxy groups can be removed without untoward effect on affinity, and relocation of the methoxy substituents actually enhances affinity by as much as an order of magnitude. None of the compounds displayed more than 20-fold selectivity for 5-HT(2A) over 5-HT(2C) receptors. In addition, several were demonstrated to act as 5-HT(2A) partial agonists. As such, the results of this study suggest that the structure-affinity relationships of phenylalkylamines as 5-HT(2A) ligands now be reinvestigated in greater detail.

Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors.

A large series of beta-carbolines was examined for their ability to bind at [3H]agonist-labeled 5-HT(2A) serotonin receptors. Selected beta-carbolines were also examined at 5-HT(2C) serotonin receptors, 5-HT(1A) serotonin receptors, dopamine D(2) receptors, and benzodiazepine receptors. Indolealkylamines and phenylisopropylamines were also evaluated in some of these binding assays. The beta-carbolines were found to bind with modest affinity at 5-HT(2A) receptors, and affinity was highly dependent upon the presence of ring substituents and ring saturation. The beta-carbolines displayed little to no affinity for 5-HT(1A) serotonin receptors, dopamine D(2) receptors and, with the exception of beta-CCM, for benzodiazepine receptors. Examples of beta-carbolines, indolealkylamines (i.e. N,N-dimethyltryptamine analogs), and phenylisopropylamines have been previously shown to produce common stimulus effects in animals trained to discriminate the phenylisopropylamine hallucinogen DOM (i.e. 1-(2, 5-dimethoxy-4-methylphenyl)-2-aminopropane) from vehicle. Although the only common receptor population that might account for this action is 5-HT(2A), on the basis of a lack of enhanced affinity for agonist-labeled 5-HT(2A) receptors, as well as on their lack of agonist action in the PI hydrolysis assay, it is difficult to conclude that the beta-carbolines behave in a manner consistent with that of other classical hallucinogens.

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised ternary complex mechanism at serotonin 5-HT(2A) and 5-HT(2C) receptors.

The ternary complex model as applied to G-protein coupled receptors (GPCR) predicts that an agonist binds with low affinity (K(L)) to the free receptor (R), leading to an agonist/receptor/G-protein complex. This ternary complex displays high agonist affinity (K(H)), resulting in signal transduction. Classical dogma states that the ratio K(L)/K(H) predicts intrinsic activity of drugs: the higher the ratio the higher the intrinsic activity. This model was based on studies in which K(L) and K(H) were indirectly determined by computer analyses of antagonist radioligand binding data. In order to investigate the relationship of K(L), K(H), and intrinsic activity for agonists at 5-HT(2A) and 5-HT(2C) receptors, we utilized (3)H-agonist and (3)H-antagonist radioligands to directly determine K(H) and K(L). Comparisons of the log K(L)/K(H) ratios and intrinsic activities of drugs for stimulating intracellular phosphatidylinositol (PI) hydrolysis revealed a strong correlation for 5-HT(2A) (r(2) = 0.92) and 5-HT(2C) (r(2) = 0.96) receptors. The data were fit to computer simulations based on the original ternary complex model and the revised ternary complex model in which an activated state of the receptor (R*) exists in equilibrium with the resting state of the receptor (R). Data produced for both 5-HT(2A) and 5-HT(2C) receptors were better-fitted to a revised ternary complex model, rather than the classical ternary complex model. These data support a revised model for the molecular events coupling GPCR to activation of G-proteins and indicate that a strong correlation between the K(L)/K(H) ratio and intrinsic activity for agonist action at GPCR is consistent with the existence of R*.

Beta-blocker selectivity at cloned human beta 1- and beta 2-adrenergic receptors.

The ratio between the affinities of beta-blockers for the beta2- and beta1-receptors is often used to predict the cardioselectivity and the potential consequences of blocking beta2-receptor-mediated effects of adrenergic receptor blockers. These ratios have been traditionally determined using various in vitro models of beta2 and beta1-receptor antagonist activity, including isolated organ preparations and radioligand binding in tissues from various species. The data from these studies, while useful, are complicated by the use of different preparations, techniques, and nonhuman models. Recombinant cell lines expressing human beta2 and beta1 receptors have been developed, allowing for the direct comparison of the affinities of the beta-blockers for the beta2 and beta1 receptors under identical conditions, and allowing a precise determination of the beta1-receptor selectivity of the beta-blockers. Bisoprolol, atenolol, propranolol, betaxolol, metoprolol, carvedilol, and ICI 118, 551 were compared for their beta-receptor selectivity using membranes prepared from recombinant cells selectively expressing human beta2 and beta1 receptors. Bisoprolol was found to have the highest selectivity for the beta1 receptor, displaying a beta2/beta1 ratio of 19 (a 19-fold higher affinity for the beta3 receptor than for the beta2 receptor). Atenolol, metoprolol, and betaxolol displayed lower, selectivity for the beta1 receptor, whereas propranolol and carvedilol displayed no significant beta-adrenergic selectivity. ICI 118,55 was selective for the beta2 receptor. The equilibrium dissociation constants of the beta-blockers for the beta1 and beta2 receptors were generally similar to previously reported values. The affinity ratios were also generally similar to previously reported values.

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding.

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.

Investigation of hallucinogenic and related beta-carbolines.

Certain beta-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 beta-carbolines at rat 5-HT2A and 5-HT2C receptors. Affinities (Ki values) of the beta-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 beta-carboline harmaline (3.0 mg/kg, i.p.) from vehicle using a VI-15s schedule of reinforcement. This represents the first time a hallucinogenic beta-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 beta-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.

Creation of a constitutively activated state of the 5-hydroxytryptamine2A receptor by site-directed mutagenesis: inverse agonist activity of antipsychotic drugs.

Single amino acid mutations in the third intracellular loop, as well as other domains of G protein-coupled receptors, have been shown to confer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an amino acid mutation can confer dramatic alterations in the 5-hydroxytryptamine2A (5-HT2A) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine (C322R). Transient expression of the mutant receptors revealed properties associated with constitutive activity. Radioligand binding studies revealed an increase in 5-HT affinity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for stimulation of inositol phosphate production increased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cells expressing native 5-HT2A receptors. Basal levels of inositol phosphate stimulated by C322K receptors represented 48% of total inositol phosphate production stimulated by native receptors in the presence of 10 microM 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperidol, loxapine and risperidone) displayed inverse agonist activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid 322 in the 5-HT2A receptor plays an important role in maintaining the inactive conformation and provide further evidence that amino acid mutations can produce profound alterations in G protein-coupled receptor activity.

Benzylimidazolines as h5-HT1B/1D serotonin receptor ligands: a structure-affinity investigation.

Benzylimidazolines may represent a class of 5-HT1D ligands that has yet to be exploited. On the basis of a previous report that the 2-(substituted-benzyl)imidazoline alpha-adrenergic agonist oxymetazoline (8) binds with high affinity at calf brain 5-HT1D receptors, we explored the structure-affinity relationships of a series of related derivatives. Each of the aromatic substituents was removed and then reinstated in a systematic manner to determine the influence of the individual substituents on binding. It was found that all of the aromatic substituents of 8 act in concert to impart high affinity. However, although the 3-hydroxy group could be removed without significantly reducing affinity for h5-HT1D (i.e., human 5-HT1Dalpha) receptors, this modification reduced h5-HT1B (i.e., human 5-HT1Dbeta) receptor affinity by nearly 50-fold. The 2, 6-dimethyl groups also contribute to binding but seem to play a greater role for h5-HT1B binding than h5-HT1D binding. With the appropriate structural modifications, several compounds were identified that display 20- to >100-fold selectivity for h5-HT1D versus h5-HT1B receptors. Preliminary functional data suggest that these compounds behave as agonists. Given that 5-HT1D agonists are currently being explored for their antimigraine action and that activation of h5-HT1B receptors might be associated with cardiovascular side effects, h5-HT1D-selective agents may offer a new lead for the development of therapeutically efficacious agents.

Agonist activity of LSD and lisuride at cloned 5HT2A and 5HT2C receptors.

Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT2C receptor and an agonist at the 5HT2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT2A and 5HT2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT2A receptors with EC50 values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT2C receptors with EC50 values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT2A and 5HT2C receptors in recombinant cells. As agonist activity at brain 5HT2A receptors has been associated with hallucinogenic activity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD.

Creation of a constitutively activated state of the 5-HT2A receptor by site-directed mutagenesis: revelation of inverse agonist activity of antagonists.

Constitutively active GPCR have revealed novel properties of drugs that exhibit classical competitive antagonism at the native forms of GPCR. These drugs reverse basal levels of constitutive activity, indicating that they have inverse agonist activity. We were interested in determining if competitive antagonists of the native 5-HT2A receptor, in particular, antipsychotic drugs, exhibit inverse agonist activity at the constitutively active 5-HT2A receptor. All of the drugs tested reduced basal IP production of constitutively active 5-HT2A receptors, indicating that they all exhibited inverse agonist activity. Risperidone and ketanserin produced the greatest inhibition of basal IP production resulting in a reduction of basal activity in the C322K mutant receptor of 82% and 80%, respectively. Antipsychotic drugs display inverse agonist activity, indicating that stabilization of the inactive conformation of the 5-HT2A receptor may be a key component of their mechanism of action.

Pharmacological characterization of the constitutively activated state of the serotonin 5-HT2C receptor.

Previous studies from our laboratory have shown that the 5-HT2C serotonin receptor can be rendered constitutively active by changing amino acid 312 (third intracellular loop) from serine to lysine (S312K). In the present study, detailed radioligand binding analyses were performed to characterize the constitutively activated state of S312K mutant receptors. All agonists tested displayed high affinity for both [3H]5-HT and [3H]mesulergine binding to S312K receptors, but displayed low affinity for [3H]mesulergine binding to native 5-HT2C receptors. [3H]5-HT labeled the same total number of S312K binding sites as [3H]mesulergine. 5-HT2C antagonists inhibited S312K basal inositol phosphate production. These results suggest that S312K receptors mimic the active conformation of native 5-HT2C receptors and provide a good model system for evaluating drugs for inverse agonist activity. Also, S312K receptors may represent a new system for screening 5-HT2C agonist activity by comparing [3H]mesulergine binding to native and S312K mutant receptors.

Activating mutations of the serotonin 5-HT2C receptor.

Site-directed mutagenesis was performed to create a mutant serotonin 5-HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [3H]mesulergine-labeled 5-HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5-HT and [3H]mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [3H]5-HT binding. These results indicate that serine --> lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.

2-(1-Naphthyloxy)ethylamines with enhanced affinity for human 5-HT1D beta (h5-HT1B) serotonin receptors.

Although the beta-adrenergic antagonist propranolol (1) binds at rodent 5-HT1B serotonin receptors, it displays low affinity (Ki > 10,000 nM) for its species homologue 5-HT1D beta (i.e., h5-HT1B) receptors. The structure of propranolol was systematically modified in an attempt to enhance its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group, shortening of the O-alkyl chain from three to two methylene groups, and variation of the terminal amine substituent resulted in compounds, such as N-monomethyl-2-(1-naphthyloxy)-ethylamine (11; Ki = 26 nM), that display significantly higher h5-HT1B affinity than propranolol. Compound 11 was shown to bind equally well at human 5-HT1D alpha (h5-HT1D) receptors (Ki = 34 nM) and was further demonstrated to possess h5-HT1B agonist character in an adenylate cyclase assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT1D receptor agonists.