Simple compounds with high pharmacologic potential: beta-carbolines. Origins, syntheses, biological properties

We reviewed the origins, the synthetic pathways and the biological properties of beta-carbolines, the condensation products of tryptophan and indole alkylamines with aldehydes. They were found in many plants, some of which have been used as hallucinogens. They also occur as minor constituents in tobacco smoke. In mammalian body, beta-carboline derivatives occur normally in plasma, platelets and urine, moreover it seems that some are formed in human body after alcohol intake. Due to interesting biological effects described in recent years (inhibition of monoamine oxidase, binding to benzodiazepine receptors, comutagenic and carcinogenic properties, 5-hydroxy tryptamine uptake inhibition), many attempts were made to prepare beta-carbolines starting from various indole derivatives. We reviewed the published methods up to 1975 and summarized the main patents related with pharmacological properties of synthetic beta-carbolines.     

Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites

The affinity of beta-carbolines, which may be formed in the body, to benzodiazepine and opiate receptors was studied by measuring their ability to inhibit the binding of [3H]-flunitrazepam and [3H]-dihydromorphine on rat brain synaptosomal membranes. All "aromatized" beta-carbolines studied (norharmane, harmane and 6-methoxyharmane) inhibited the specific binding of [3H]-flunitrazepam in micromolar concentrations, dihydro-beta-carbolines (6-methoxyharmalan, harmalol) were less potent, while all tetrahydro-beta-carbolines showed very low affinity. 6-Hydroxytetrahydroharmane, which is formed by condensation 5HT with acetaldehyde, inhibited [3H]-dihydromorphine binding in micromolar concentration, while norharmane and tetrahydro-beta-carbolines without OH-group showed little affinity. beta-Carbolines are the most potent known natural benzodiazepine receptor ligands. Because they are formed after alcohol drinking, their effects on benzodiazepine and opiate receptors may be connected with alcohol dependence although some beta-carbolines may inhibit 5HT uptake in still lower concentrations.     

Inhibition of Brain Type A Monoamine Oxidase and 5-Hydroxytryptamine Uptake by Two Amphetamine Metabolites, p-Hydroxyamphetamine and p-Hydroxynorephedrine

Two amphetamine metabolites, p-hydroxyamphetamine (p-OHA) and p-hydroxynorephedrine (p-OHN), selectively inhibited the A form of monoamine oxidase (MAO) in rat and mouse forebrain homogenates. Of these two metabolites, p-OHA inhibited MAO-A more strongly than p-OHN. This MAO-A-selective inhibition by p-OHA or p-OHN was found to be competitive with respect to deamination of its substrate, 5-hydroxytryptamine (5-HT). The degree of MAO-A inhibition was not changed by 90 min of preincubation of the enzyme preparations with either metabolite, and the activity inhibited by p-OHA after the preincubation recovered completely to the control level after repeated washing. Uptake of 5-HT or dopamine into mouse forebrain synaptosomes was highly reduced by both p-OHA and p-OHN. Both metabolites were more potent in reducing dopamine uptake than in reducing 5-HT uptake. In reduction of 5-HT and of dopamine uptake, p-OHA was more potent than p-OHN. These results indicate that p-OHA is a more selective inhibitor of brain MAO-A activity and 5-HT uptake than its subsequent metabolite, p-OHN. These two actions of p-OHA might, together with possible 5-HT efflux into the synaptic cleft, greatly contribute to head twitch, a brain 5-HT-mediated animal behavior induced by p-OHA.     

Human monoamine oxidase is inhibited by tobacco smoke: beta-carboline alkaloids act as potent and reversible inhibitors

Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two beta-carboline alkaloids, norharman (beta-carboline) and harman (1-methyl-beta-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that beta-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K(i)=1.2+/-0.18 microM) and MAO-B (K(i)=1.12+/-0.19 microM), and harman of MAO-A (K(i)=55.54+/-5.3nM). Beta-carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that beta-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like beta-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking.     

Selective inhibition of MAO-A in serotonergic synaptosomes by two amphetamine metabolites, p-hydroxyamphetamine and p-hydroxynorephedrine

The present study was carried out mainly to clarify whether the two amphetamine metabolites, p-hydroxyamphetamine (P-OHA) and p-hydroxynorephedrine (p-OHN) are taken up by mouse brain 5-hydroxytryptamine (5-HT) nerve terminals to inhibit type A monoamine oxidase (MAO-A) and then potentiate the abnormal behavior, head-twitch. Of the two metabolites, only intracerebroventricular p-OHA, at 80 μg/mouse, sufficient to cause a head-twitch response (HTR), appreciably inhibited MAO-A activity without affecting MAO-B activity in homogenates of the mouse striatum, hypothalamus and the rest of the forebrain; and p-OHN did not inhibit either type of MAO at the dose tested. Estimation of intra- and extrasynaptosomal MAO-A activity showed that both metabolites significantly inhibited only the intrasynaptosomal deamination of 5-HT by MAO-A with p-OHA being more potent. Taken together with our previous findings, these present results clearly indicate that p-OHA may accumulate in the 5-HT nerve terminals through the uptake system, and concomitantly inhibit MAO-A activity. These actions of p-OHA may increase intraneuronal 5-HT levels and then potentiate 5-HT release to cause interaction with the post-synaptic 5-HT receptors.     

Antagonism of Benzodiazepine Binding in Brain by Antilirium, Benzyl Alcohol, and Physostigmine

Abstract: Antilirium®, which contains eserine (physostigmine) and benzyl alcohol, is effective in reversing diazepam-induced sleep in man and is capable in vitro of inhibiting the binding of labeled benzodiazepine to both rat and human brain homogenates in a dose-dependent manner. We have examined the constituents of Antilirium and report that both benzyl alcohol and eserine inhibit [³HI-diazepam binding to rat brain in a dose-dependent manner. A major portion of the inhibition of binding found with Antilirium is accounted for by benzyl alcohol. Scatchard analysis of inhibition of benzodiazepine binding by benzyl alcohol reveals loss of binding sites and change in equilibrium dissociation constant. Methanol, ethanol, and butanol did not inhibit benzodiazepine binding. The inhibition by benzyl alcohol may be specific since there was no inhibition of labeled ligand binding to the γ-aminobutyric acid, opiate, muscarinic acetylcholine, or β-adrenergic receptors. The other constituent, eserine, is a competitive inhibitor. While eserine is a more potent inhibitor at the benzodiazepine receptor than is benzyl alcohol, it is also much less specific. Eserine inhibited binding of labeled ligand to the γ-aminobutyric acid, opiate, and muscarinic acetylcholine receptors. The inhibition of benzodiazepine binding to brain in vitro by Antilirium and its constituents, eserine and benzyl alcohol, may be the explanation, at least in part, for the reversal by Antilirium of diazepam-induced narcosis in viva, without postulating a cholinergic mechanism for the in vivo effect.     

Fine mapping of a sedative-hypnotic drug withdrawal locus on mouse chromosome 11

We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative-hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28-cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3-cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABA(A) receptor, which is implicated as a pivotal component in sedative-hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative-hypnotic drugs, encodes a unique GABA(A) receptor gamma2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for alpha1 subunit containing GABA(A) receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABA(A) receptor gamma2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative-hypnotic physiological dependence and associated withdrawal episodes.     

Benzodiazepine Enhancement of γ-Aminobutyric Acid-Mediated Chloride Ion Flux in Rat Brain Synaptoneurosomes

Benzodiazepine agonists such as Ro 11-6896 [B10(+)], diazepam, clonazepam, and flurazepam were found to enhance muscimol-stimulated 36Cl- uptake into rat cerebral cortical synaptoneurosomes. The rank order of potentiation was B10(+) greater than diazepam greater than clonazepam greater than flurazepam. These benzodiazepines had no effect on 36Cl-uptake in the absence of muscimol. Further, the inactive enantiomer, Ro 11-6893 [B10(-)], and the peripheral benzodiazepine receptor ligand Ro 5-4864 did not potentiate muscimol-stimulated 36Cl- uptake at concentrations up to 10 microM. In contrast, the benzodiazepine receptor inverse agonists ethyl-beta-carboline-3-carboxylate and 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylic acid methyl ester inhibited muscimol stimulated 36Cl- uptake. Benzodiazepines and beta-carbolines altered the apparent K0.5 of muscimol-stimulated 36Cl- uptake, without affecting the Vmax. The effects of both benzodiazepine receptor agonists and inverse agonists were reversed by the benzodiazepine antagonists Ro 15-1788 and CGS-8216. These data further confirm that central benzodiazepine receptors modulate the capacity of gamma-aminobutyric acid receptor agonists to enhance chloride transport and provide a biochemical technique for studying benzodiazepine receptor function in vitro.     

Modulation of γ-Aminobutyric AcidA Receptor-Operated Chloride Channels by Benzodiazepine Inverse Agonists Is Related to Genetic Differences in Ethanol Withdrawal Seizure Severity

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.     

Discriminative and aversive properties of beta-carboline-3-carboxylic acid ethyl ester, a benzodiazepine receptor inverse agonist, in rhesus monkeys

Rhesus monkeys were trained to discriminate injections of saline from those of beta-carboline-3-carboxylic acid ethyl ester (beta-CCE), a compound that binds to the benzodiazepine receptor, but often has actions opposite to those of the benzodiazepines. A benzodiazepine agonist midazolam and low doses of a specific benzodiazepine antagonist, Ro 15-1788, reversed the discriminative effects of beta-CCE. Higher doses of Ro 15-1788 produced stimulus effects similar to beta-CCE. In a separate experiment, monkeys responded to terminate intravenous infusions of beta-CCE, but not midazolam. This aversive effect of beta-CCE was reversed by Ro 15-1788. The behavioral effects of beta-CCE in these non-human primates are consistent with other data that have shown it to act on benzodiazepine receptors, and support the hypothesis that beta-CCE can be considered an inverse agonist at this receptor.     

Electrophysiological actions of benzodiazepines

Electrophysiological investigations have revealed that benzodiazepines, applied either locally or systemically, reduce central nervous system excitability. The studies summarized here indicate that this depression of excitability by benzodiazepines is a result of an increase in gamma-aminobutyric acid (GABA) mediated inhibition. This increase in inhibition may result from benzodiazepines increasing the activity of some GABAergic neurons and also from a modulatory action of benzodiazepines on GABA actions at some postsynaptic receptor sites. The modulatory action is observed with doses of benzodiazepines that do not cause any direct effects on neuronal excitability or membrane polarization. Specificity tests indicate that benzodiazepines do not enhance inhibition mediated by glycine or monoamines such as norepinephrine or serotonin. Results of experiments with a convulsant benzodiazepine compound, which causes a specific reduction in GABA-mediated inhibition, are also presented, The data are discussed in terms of a model in which the benzodiazepine receptor, the GABA receptor, and the chloride ionophore are functionally linked. Furthermore, it is proposed that some postsynaptic actions of GABA may be continually regulated by the occupancy of a benzodiazepine receptor, and that occupancy of the benzodiazepine receptor may be permissive for the GABA-elicited increase in chloride ion permeability.     

Modification of the effects of naloxone in morphine-dependent mice

Mice were rendered dependent on morphine by mixing morphine with their food (2 mg/g) for three days. Increasing doses of naloxone precipitated dose-dependent withdrawal reactions such as weight loss and jumping. These withdrawal reactions were antagonized by morphine pretreatment. Effects of morphine, such as increased locomotor activity, inhibition of intestinal transport, and analgesia were antagonized by naloxone in both non-dependent and dependent subjects. The antagonist actions of naloxone were increased in dependent subjects; lower doses of naloxone were sufficient to antagonize effects of morphine. The present results confirm earlier studies indicating that precipitation of withdrawal can be antagonized by morphine pretreatment suggesting that withdrawal reactions are due to actions of naloxone at the same receptor at which opioid agonists act. The increased antagonist potency of naloxone in dependent subjects extends earlier results obtained with analgesic effects to several other agonist effects of morphine and is consistent with the interpretation that exposure to an opioid agonist induces a change in the conformation of opioid receptors.     

Serotonin-mediated protein carbonylation in the right heart

Pulmonary hypertension is a devastating disease, which leads to right heart failure. Serotonin (5-HT) plays important roles in the pathogenesis of pulmonary hypertension and pulmonary vascular remodeling. The role of 5-HT in right heart failure, however, is unknown. Since oxidative stress may mediate heart failure, the present study examined the effects of 5-HT on protein oxidation in the adult rat right heart ventricle. Treatment of perfused isolated hearts with 5-HT resulted in the promotion of protein carbonylation, specifically in the right ventricle, but not in the left. While no differences between right and left ventricular antioxidant enzymes and 5-HT receptors/transporter were detected, monoamine oxidase A (MAO-A) expression and activity were found to be lower in the right ventricle compared to the left. These results indicate that differences in neither the reactive oxygen species (ROS) scavenging ability, 5-HT membrane signaling capacity, nor MAO-dependent production of hydrogen peroxide are responsible for varied 5-HT-mediated protein carbonylation in right and left ventricles. Rather, lower MAO-A in the right heart might preserve cytosolic 5-HT which triggers other mechanisms for ROS production. Consistently, inhibition of MAO-A resulted in the promotion of protein carbonylation. We propose that low MAO-A, thus reduced degradation of 5-HT, increases the intracellular 5-HT activity in the right ventricle, leading to the promotion of protein carbonylation.     

Flumazenil selectively prevents the increase in alpha(4)-subunit gene expression and an associated change in GABA(A) receptor function induced by ethanol withdrawal

The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.     

GABA-benzodiazepine interactions: physiological, pharmacological and developmental aspects

Many of the pharmacological actions of the benzodiazepines can be attributed to their actions on gamma-aminobutyric acid (GABA) systms in the brain. Electrophysiological studies on dorsal raphe neurons indicate that the benzodiazepines act postsynaptically to potentiate GABAergic inhibition in this midbrain nucleus. Direct binding studies have shown that both in vitro and in vivo binding of [3H]diazepam to a specific high affinity benzodiazepine binding site in cerebral cortical tissue are enhanced by the direct in vitro addition of GABA and GABA agonists or by pretreatment of animals with GABA analogs and agents that elevate GABA levels in brain. Ontogenic development of [3H]diazepam binding in brain parallels the development of the sodium-independent [3H]GABA binding. The ability of GABA to enhance benzodiazepine binding is present throughout development and inversely related to age. These data suggest that there is a functionally significant interaction between the benzodiazepines and GABA throughout development and at maturity. A model is proposed to relate these interactions to conformational changes in a benzodiazepine/GABA/Cl- ionophore complex.     

GABA-benzodiazepine receptor complex and drug actions

This short review describes the benzodiazepine receptors, their interplay with GABAergic transmission and chloride ionophore, the search for endogenous ligands, and the drug responses that can be evoked through these receptors. Benzodiazepine receptors offer a unique pathway through which opposite drug actions e.g., anxiogenic and anxiolytic effects can be exerted, and these actions can be inhibited with competitive receptor antagonists. The most plausible endogenous ligand for benzodiazepine receptors discovered so far, a polypeptide DBI, exerts actions opposite to those of the benzodiazepines used in clinical therapy. This has been the stimulus for a new look at the physiological role for these receptors.     

The expression and activity of monoamine oxidase A, but not of the serotonin transporter, is decreased in human placenta from pre-eclamptic pregnancies

Serotonin (5-HT) plays a pivotal role in pregnancy and a hyperserotonomic condition has been documented in pre-eclampsia. We have attempted to elucidate the possible participation of 5-HT as an aetiological factor in pre-eclampsia, by estimating the activity and expression of the 5-HT transporter (SERT) and monoamine oxidase A (MAO-A) in human placenta from full term normal (NG) and severe pre-eclamptic (PES) pregnancies. Uptake of 5-[1,2-3H] hydroxytryptamine binoxalate (specific radioactivity, 30.4 Ci/mmol) was determined in placental brush border vesicles by a rapid filtration technique. 5-HT metabolism in placental homogenate was measured using a HPLC-ECD system. Expression of SERT and MAO-A was determined by Western blot, using specific antibodies against the human SERT and MAO-A in placental tissues obtained from NG and PES. Our results, showed no significant difference in 5-HT uptake between both groups. However, 5-HT metabolism was significantly lower in placental homogenates from PES than in NG placentas, with the pathological preparations showing no MAO-A activity against 5-HT during the first 60 min of incubation (87% and 5% of metabolism of 5-HT initially added, NG and PES respectively). Western blot analysis showed a similar expression of SERT in BBMV from NG and PES. However, unlike for normal pregnancies, the expression of MAO-A in placental homogenates from PES was found to be very low, or almost negligible. These findings confirm our previous results and suggest that the higher plasma free 5-HT levels observed in severe pre-eclampsia could be mainly due to a reduction in placental MAO-A expression and activity and are not limited by the expression and uptake of 5-HT into the placental tissue.     

The effects of 5-HT1B characterizing agents in the mouse elevated plus-maze

Although the serotonergic system has been implicated in the modulation of anxiety states, the specific receptor subtypes that mediate these states require clarification. The effects of drugs that act preferentially at 5-HT1B receptors were evaluated on the behavior elicited in the elevated plus-maze, an animal model of anxiety. Variations in the intensity of light affected mouse behavior in the plus-maze; lower light intensity increased the entries to and time spent on the open arm in a manner similar to that seen with stress-attenuating circumstances. Opposite effects were observed in high light-intensity, similar to effects seen under elevated stress conditions. Chlordiazepoxide produced increased entries and time spent on the open arm, whereas pentylenetetrazol (PTZ) produced opposite effects. The preferential 5-HT1B agents TFMPP and mCPP exhibited a profile similar to PTZ. The effects of TFMPP in the plus-maze were reversed by chlordiazepoxide, but not by the benzodiazepine receptor antagonist flumazenil, which suggests that this effect is not directly mediated by benzodiazepine receptors. The decreased entries and time spent on the open arm of the maze following TFMPP or mCPP administration was possibly mediated by an antagonistic action at 5-HT1B receptors, since this effect was reversed by the selective 5-HT1B agonist CGS 12066B. The present study further demonstrates the utility of mouse behavior in the elevated plus-maze as a model for identifying anxio-modulatory substances.     

Pharmacological activities of Vitex agnus-castus extracts in vitro.

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.     

Expression of serotonin receptors in bone

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.