Serotonin Binding Research Articles

De Novo Discovery of Serotonin N-Acetyltransferase Inhibitors

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.

Caveolin-1 affects serotonin binding and cell surface levels of human 5-HT 7(a) receptors

Studies using HeLa cells expressing 5-HT 7 receptors showed that detergent resistant membranous lipid rafts purified by sucrose gradient centrifugation contained 5-HT 7 receptors and caveolin-1. Caveolin-1 siRNA-mediated knockdown or serotonin (5-HT) treatment caused significant reductions of maximum [ 3H]5-HT binding to 5-HT 7 receptors and total immunoreactivity of membranous 5-HT 7 receptors in intact cells. Co-treatment with 5-HT, caveolin-1 siRNA and methyl-β-cyclodextrin had no additive effects on reducing maximum binding of [ 3H]5-HT to 5-HT 7 receptors. 5-HT-mediated reduction of [ 3H]5-HT binding to 5-HT 7 receptors was counteracted by genistein, but not by sucrose. Caveolin-1, specifically localized in cholesterol-enriched lipid rafts, appears to regulate constitutive and agonist-stimulated cell surface levels of 5-HT 7 receptors via a clathrin-independent mechanism.

Membrane organization and function of the serotonin(1A) receptor.

(1) The serotonin(1A) receptor is a G-protein coupled receptor involved in several cognitive, behavioral, and developmental functions. It binds the neurotransmitter serotonin and signals across the membrane through its interactions with heterotrimeric G-proteins. (2) Lipid-protein interactions in membranes play an important role in the assembly, stability, and function of membrane proteins. The role of membrane environment in serotonin(1A) receptor function is beginning to be addressed by exploring the consequences of lipid manipulations on the ligand binding and G-protein coupling of serotonin(1A) receptors, the ability to functionally solubilize the serotonin(1A) receptor, and the factors influencing the membrane organization of the serotonin(1A) receptor. (3) Recent developments involving the application of detergent-based and detergent-free approaches to understand the membrane organization of the serotonin(1A) receptor under conditions of ligand activation and modulation of membrane lipid content, with an emphasis on membrane cholesterol, are described.

Segregation of Platelet Aggregatory and Procoagulant Microdomains in Thrombus Formation

Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated alphaIIb beta3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active alphaIIb beta3. These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.

3H] Citalopram Binding to Serotonin Transporter Sites in Minnow Brains

Mammalian serotonin (SERT) and norepinephrine transporters (NET) are target sites for antidepressants and are affected by pesticide exposures. Herein, we examined whether golden shiner (Notemigonus crysoleucas) or fathead minnow (Pimphales promelas) SERTs and catecholamine transporters respond comparably to mammalian SERTs and NETs. We compared the pharmacological profiles of central SERT and NET binding sites of the golden shiner minnow to those of rats. Homogenate binding with the radioligand [(3)H] citalopram indicated that golden shiner SERT has a K(D) of 7 +/- 3 nM and a B(max) of 226 +/- 46 fmol/mg protein. These values are similar to those of rat cortical SERT (K(D) 1.4 +/- 0.1 nM and B(max) 240 +/- 48 fmol/mg protein). We also examined SERT binding in fathead minnow brain, and found it similar to that of the golden shiner. A putative golden shiner NET, measured using [(3)H] nisoxetine, had K(D) = 12 +/- 5 nM and B(max) = 187 +/- 49 fmol/mg protein, whereas rat hippocampal NET had K(D) = 5 +/- 2 nM and B(max) = 93 +/- 8 fmol/mg protein. Minnow SERT and NET binding is displaceable by selective reuptake inhibitors. Finally, we exposed zebrafish (Danio rerio) to the serotonin reuptake inhibiting antidepressant sertraline or the organophosphate chlorpyrifos for 21 days. After either treatment, SERT binding was reduced by 50% (n = 3-6, P < 0.05). In summary, minnow central SERT and NET express slightly lower affinity for antidepressants than rats. However, magnitudes of affinity are similar, and minnow SERT binding is decreased by chronic sertraline or chlorpyrifos administration.

Autoradiography reveals selective changes in serotonin binding in neocortex of patients with temporal lobe epilepsy

The main goal of the present study was to evaluate binding to serotonin in the neocortex surrounding the epileptic focus of patients with mesial temporal lobe epilepsy (MTLE). Binding to 5-HT, 5-HT 1A, 5-HT 4, 5-HT 7 receptors and serotonin transporter (5-HTT) in T1–T2 gyri of 15 patients with MTLE and their correlations with clinical data, neuronal count and volume were determined. Autopsy material acquired from subjects without epilepsy ( n = 6) was used as control. The neocortex from MTLE patients demonstrated decreased cell count in layers III–IV (21%). No significant changes were detected on the neuronal volume. Autoradiography experiments showed the following results: reduced 5-HT and 5-HT 1A binding in layers I–II (24% and 92%, respectively); enhanced 5-HT 4 binding in layers V–VI (32%); no significant changes in 5-HT 7 binding; reduced 5-HTT binding in all layers (I–II, 90.3%; III–IV, 90.3%, V–VI, 86.9%). Significant correlations were found between binding to 5-HT 4 and 5-HT 7 receptors and age of seizure onset, duration of epilepsy and duration of antiepileptic treatment. The present results support an impaired serotoninergic transmission in the neocortex surrounding the epileptic focus of patients with MTLE, a situation that could be involved in the initiation and propagation of seizure activity.

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC 50 values of HD-205 were 4.1, 14 and 280 nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC 50 values of 191, 230 and 840 nM at DAT, SERT and NET, respectively. Saturation binding studies with [ 125I]RTI-55 in membranes pretreated with 100 nM HD-205 showed that HD-205 significantly decreased the B max but not K D of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC 50 of HD-244 at DAT was 20 nM. [ 125I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [ 125I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.

Depletion of the lipid raft constituents, sphingomyelin and ganglioside, decreases serotonin binding at human 5‐HT7(a) receptors in HeLa cells

The localization and function of several G protein-coupled receptors, including beta-adrenergic receptors and NK 1 receptors, are regulated via lipid rafts in the plasma membrane. These domains are enriched in cholesterol, gangliosides and sphingolipids, and play an important role in regulating signal transduction in most cell types. Serotonin (5-hydroxytryptamine, 5-HT), acting via 14 different receptors, regulates as diverse effects as mood, metabolism and smooth muscle contraction. 5-HT(7) receptors are involved in the regulation of depression, circadian rhythms, thermoregulation and vasodilatation. Ligand binding and signalling via the 5-HT(7) receptor are regulated by membranous cholesterol. Here we investigated the role of sphingomyelin and gangliosides on binding of 5-HT to 5-HT(7) receptors to further examine the role of lipid raft constituents on 5-HT(7) receptor function. HeLa cells stably transfected with the human 5-HT(7) receptor were treated with Fumonisin B(1) or (+/-)-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) to reduce sphingomyelin or ganglioside levels, respectively. The effects of these treatments were investigated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) viability assay, cholesterol analysis and [(3)H]5-HT binding studies on intact cells. Treatments with 20 mum Fumonisin B(1) for 24 h or with 10 mum PDMP for 48 h had no effects of total levels if 5-HT(7) receptors, but caused significant decreases in maximum [(3)H]5-HT binding to 5-HT(7) receptors. The effects were cholesterol-independent as levels of cholesterol remained unaffected by either treatment. These data demonstrate a role for sphingomyelin and gangliosides in regulating binding of [(3)H]5-HT to 5-HT(7) receptors. These observations further strengthen that actions of 5-HT via 5-HT(7) receptors are dependent upon lipid raft integrity.

Functional activation by central monoamines of human dopamine D 4 receptor polymorphic variants coupled to GIRK channels in Xenopus oocytes

We studied the functional activation of different polymorphic variants of the human dopamine D 4 receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D 4 receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D 4 receptor variants coupled to oocyte G i/o proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D 4 receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC 50 = 1 nM) than on D4.4 (EC 50 = 5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D 4 receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D 4 receptor variants (EC 50 NA = 50 nM, EC 50 5-HT = 1.5 μM). All monoamine effects were blocked by the specific dopaminergic D 4 antagonist (S)-(−)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D 4 receptors and related monoamine receptors revealed that dopamine D 4 receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D 4 receptors can be pharmacologically stimulated by any the three major central monoamines.

Analysis of Antiemetic Effect of Various Dosage Regimens of Azasetron Hydrochloride Based on 5-HT&lt;sub&gt;3&lt;/sub&gt; Receptor Occupancy of Serotonin

Antineoplastic drugs have been shown to exert direct effects on the gut and induce the release of serotonin from the enterochromaffin cells of small intestinal mucosa. It is thought that released serotonin stimulates vagal afferent fibers through 5-HT3 receptors located in the vagal afferent terminals in the gastrointestinal tract and initiates sensory signals to the area postrema and the emetic center, thereby initiating nausea and vomiting. A 5-HT3 antagonist competitively inhibits serotonin at its specific binding sites, 5-HT3 receptors, and thereby elicits an antiemetic effect. Therefore 5-HT3 receptor occupancy of serotonin may be an appropriate indicator of the antiemetic activity of 5-HT3 antagonists. We analyzed 5-HT3 receptor occupancy of serotonin by integrating pharmacokinetic and receptor-binding kinetic parameters based on the receptor occupancy theory to compare the strength of the antiemetic effects of three dosage regimens of azasetron hydrochloride. The inhibitory effects on the binding of serotonin to 5-HT3 receptor of regimen 2 (an intravenous bolus injection of 5 mg of azasetron hydrochloride before and 8 h after chemotherapy) and regimen 3 (an intravenous bolus injection of 2.5 mg followed by 7.5 mg continuous intravenous infusion for 24 h) were longer-lasting than those of regimen 1 (an intravenous bolus injection of 10 mg before the start of chemotherapy). Furthermore, a positive relationship was found between the time of inhibitory effects on the binding of serotonin to 5-HT3 receptor and antiemetic effects of azasetron hydrochloride. From these results, dosage regimens 2 and 3 were considered to be more effective in the long term than regimen 1 in prophylaxis of nausea and vomiting induced by cisplatin.

Another new theory explaining the cause of SIDS

A great number of theories explaining the cause of the Sudden Infant Death Syndrome (SIDS) have been published during the last decades, many in so-called `high-impact' journals. In fact, few diseases in paediatrics have inspired so many scientists in various fields to solve the enigma why apparently healthy infants suddenly die. A new study was recently published in JAMA by Paterson et al. (1). The authors reported that victims of SIDS have a decreased binding of serotonin in some important brain nuclei involved in sleep and respiration. The reduced receptor binding density was particularly seen in the male SIDS cases. This finding confirms a previous observation by Ozawa et al. (2). Furthermore, Paterson et al. found a significantly higher serotonin neuron density in medullary nuclei involved in homeostatic function. These findings are very thought-provoking and also fit into the well-established finding that sleeping in prone position increases the risk. Infants who lie with the face down towards the mattress may develop desaturation and carbon dioxide retention. Normally, this should result in arousal and awakening of the infant, but deficiencies involving serotonin containing nuclei may hazard this mechanism. It is interesting that serotonin has been proposed to play a crucial role in the CO2-sensitivity (3). The finding of more marked disturbances of the serotonin system in the male SIDS cases also fit with the higher incidence of SIDS among boys. Furthermore, Weese-Mayer et al. have demonstrated that victims of SIDS often has a mutation of the PHOX2b gene, which is involved in the encoding of the serotonin synthesizing enzymes (4). This was mainly found in Afro-American infants who have a high risk of dying of SIDS. The findings are novel and of considerable interest to understand the mechanisms causing SIDS. However, the limitations of this study have also been pointed out in an Editorial in JAMA (5). The size of the study group is relatively small, with only 31 index infants and 10 control cases. The main problem is that one third of the index cases were ex-preterm infants. Although prematurity is a well-known risk factor for SIDS it cannot be excluded that the differences between the SIDS victims and the controls may be due to immaturity. Only one of the ten controls was born preterm. The finding that the expression of neurotransmitters and their receptors may be disturbed in SIDS victims is not new. The same research group has earlier reported changes of muscarinic receptors (6). Neuropeptides like substance P have also been found to be different in SIDS victims (7). Furthermore, nicotinic receptors may also be involved, since maternal smoking is one of the most important risk factors causing SIDS (8). The current findings were published in a high-impact journal and aroused a lot of media attention and were presented in the CNN news nearly every hour on the day they were published, like many other new hypotheses on SIDS (Fig. 1). This can be compared with the first reports suggesting the importance of putting the baby in a back position instead of a prone position (9-11). It took some time before they attracted attention, resulting in the most successful prevention of SIDS. Some theories on the cause of SIDS published in the New England Journal of Medicine.

Gingerol derivatives modulate T-cell receptor mediated effects in a whole blood assay and in T-cells via 5-HT1A receptor-dependent mechanisms

The rhizome of ginger (Zingiber officinale Roscoe) is widely used for digestive complaints, emesis, as well as inflammatory diseases, such as arthritis. Gingerol-derivatives have been reported to inhibit prostaglandin production, to indirectly modulate 5-HT3 receptor signalling, and also to influence calcium homeostasis. In order to assess the immunomodulatory potential of ginger we have profiled the effects of different ginger extracts in differentially stimulated whole blood. The model is more stable than assays with purified cells and the read-out is meaningful with regard to physiological function. Our experiments show that different ginger extracts (10–50µg/ml) and isolated pungent compounds specifically inhibit cytokine production in human whole blood after treatment with CD3 antibody, indicating a T-cell mediated process. Ethanolic and CO2 ginger extracts (10–50µg/ml) significantly inhibited CD3-stimulated Ca2+ release from intracellular stores in Jurkat T-cells, whereas a series of pure compounds showed similar, stimulatory, or no effects. To reduce these findings to a possible common denominator we postulated an effect on serotonin receptors as one possible mechanism. Serotonin can reach µM concentrations in whole blood and is known to be an important modulator of the immune response. Binding studies on the human 5-HT1A and 5-HT3 receptors led to the identification of nine compounds (8-; 10-gingerol, 6-; 8-; 10-shogaol, 1-dehydro-6-, 8-; and 10-gingerdione, as well as 6-dihydroparadol) which showed selective binding affinity to the serotonin binding site in 5HT1A receptors (Ki values=3 to 20µM). Moreover, ginger extracts inhibited the stimulatory effect of serotonin on CD3-stimulated calcium release in Jurkat cells. This is the first report on the immunomodulatory effects of gingerol-derivatives mediated, at least in part, via 5HT1A receptor interaction.

Exploring the binding of serotonin to the 5-HT3 receptor by density functional theory.

The 5-HT3 receptor is a typical ligand-gated ion channel of the Cys-loop superfamily, which is activated by binding of serotonin (5-HT). Models of the binding site of this protein reveal potential interactions between 5-HT and Tyr143, Tyr153, and Tyr234. Here we describe a series of ab initio calculations, based on density functional theory, to assess the effects of mutating these tyrosine residues on the binding of 5-HT. A series of mutations to these tyrosines, previously studied experimentally, were tested, and the binding energies compared with the available experimental data. Our results show that Tyr153 could form a hydrogen bond with the tertiary amine of 5-HT, and that mutation in this location revealed binding energies broadly in line with experimentally determined EC50s. Tyr143 could also form a hydrogen bond, but as EC50s do not relate to binding energies, it is unlikely that such a bond is formed here. Tyr234 is quite distinct in that it may interact with 5-HT via a mixed hydrogen bond/cation-pi interaction.

A Role for Inflammatory Mediators in the Induction of Immunoregulatory B Cells

UV exposure suppresses the immune response to a variety of microbial, fungal, and viral Ags. In addition, UV radiation is a complete carcinogen and the immune suppression induced by UV radiation is a major risk factor for skin cancer induction. In this study, we examined the mechanisms underlying the induction of immune suppression and tolerance induction by UV radiation. Transferring lymph nodes cells from UV-irradiated, FITC-sensitized mice into normal recipients transferred immune tolerance. Contrary to expectations, the cell responsible was an FITC(+), IL-10-secreting, CD19(+), B220(+) B cell. Because the lipid mediator of inflammation, platelet-activating factor (PAF) is released by UV-irradiated keratinocytes and is essential for the induction of immune suppression, we determined its role in tolerance induction. When UV-irradiated mice were injected with PCA 4248, a selective PAF receptor (PAFR) antagonist, transfer of tolerance was suppressed. However, immune suppression was not transferred when FITC(+) cells from the draining lymph nodes of UV-irradiated, PAFR-deficient donor mice were injected into the recipients. Because PCA 4248 also blocks serotonin receptor binding, we measured the effect that blocking both serotonin and PAFR binding has on the transfer of immune suppression. Only when both PAF and serotonin binding were blocked could we inhibit tolerance induction. These data identify a novel function for PAF and serotonin in modulating immune function, the activation of immunoregulatory B cells.

Cholesterol depletion reduces serotonin binding and signaling via human 5-HT 7(a) receptors

Lipids, including cholesterol, are critical components of the cell membrane where they are enriched in microdomains, lipid rafts, which organize and concentrate receptors and intracellular proteins involved in signal transduction. The present study examined the effects of cholesterol depletion on serotonin (5-HT) binding and signaling via 5-hydroxytryptamine 7 (5-HT 7) receptors in stably transfected HeLa cells. Immunohistochemical, ligand-binding and biotinylation experiments demonstrated that the studied cells expressed high levels of 5-HT 7 receptors at their surface with a pharmacological profile resembling 5-HT 7 receptors in native tissue. Depletion of cholesterol, by combined treatment with mevastatin, fumonisin B 1 and mevalonate or methyl-β-cyclodextrin (MβCD), caused highly significant reductions in the B max values of [ 3H]5-HT- and [ 3H]-( R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol ([ 3H]SB269970)-binding to 5-HT 7 receptors. Cholesterol depletion also reduced the total level of 5-HT 7 receptor protein determined by Western blot analysis. None of the examined treatments affected the affinity of [ 3H]5-HT- or [ 3H]SB269970-binding to 5-HT 7 receptors. Treatment with serotonin caused strong inductions in the phosphorylation states of Ser 63-ATF-1 and Ser 133-CREB. These effects of serotonin on signal transduction were significantly counteracted by pre-treatment with cholesterol synthesis inhibitors. Altogether, the present study demonstrates that cholesterol depletion decreases binding of both agonist and antagonist radioligands to 5-HT 7 receptors and counteract 5-HT 7 receptor-mediated intracellular signaling.

Am5‐HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera)

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT(7) receptor family. Expression of the Am5-HT(7) receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT(7) is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC(50) = 1.1-1.8 nm). The Am5-HT(7) receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Mode of action of gingerols and shogaols on 5-HT 3 receptors: Binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum

Ginger (rhizomes of Zingiber officinale) has been shown to exert potent anti-emetic properties, but its mode of action has not yet been elucidated. Among its active constituents, [6]-, [8]- and [10]-gingerol as well as [6]-shogaol were shown in different in vivo studies to be at least partly responsible for the drug's anti-emetic properties. In an attempt to gain more insight into the mode of action of these compounds, three different in vitro models were used to investigate their effects on 5-HT 3 receptors (serotonin receptor subtype) in more detail: [ 14C]guanidinium influx into N1E-115 cells which express 5-HT 3 receptors, isotonic contractions of the isolated guinea-pig ileum and equilibrium competition binding studies using a radioactively labeled 5-HT 3 receptor antagonist ([ 3H]GR65630) (3-(5-methyl-1 H-imidazol-4-yl)-1-(1-methyl-1 H-indol-3-yl)-1-propanone). All four compounds inhibited the [ 14C]guanidinium influx through 5-HT 3 receptor channels as well as contractions of the guinea-pig ileum induced by SR57227A ((4-amino)-(6-chloro-2-pyridyl) l-piperidine hydrochloride), a highly selective 5-HT 3 receptor agonist. Both effects were concentration-dependent, with the following order of potency for both models: [6]-shogaol ≥ [8]-gingerol > [10]-gingerol ≥ [6]-gingerol. All compounds showed also weak anticholinergic and antineurokininergic activities in the guinea-pig ileum (acetylcholine and substance P are mediators of the 5-HT 3 receptor effect). The vanilloid receptor did not seem to be involved derived from experiments using capsazepine. None of the tested ginger substances, however, was able to displace [ 3H]GR65630 from its binding site (5-HT 3 receptor) neither on intact N1E-115 cells nor on the purified membranes of HEK-293 cells over-expressing the h5-HT 3 receptor. It may be concluded that [6]-, [8]-, [10]-gingerol and [6]-shogaol exert their anti-emetic effect at least partly by acting on the 5-HT 3 receptor ion-channel complex, probably by binding to a modulatory site distinct from the serotonin binding site. This may include indirect effects via receptors in the signal cascade behind the 5-HT 3 receptor channel complex such as substance P receptors and muscarinic receptors; this needs further investigation since ginger is effective against motion sickness which is cured by some vanilloids and by anticholinergics such as scopolamine.

Structure and Variation of Three Canine Genes Involved in Serotonin Binding and Transport: The Serotonin Receptor 1A Gene (htr1A), Serotonin Receptor 2A Gene (htr2A), and Serotonin Transporter Gene (slc6A4)

Aggressive behavior is the most frequently encountered behavioral problem in dogs. Abnormalities in brain serotonin metabolism have been described in aggressive dogs. We studied canine serotonergic genes to investigate genetic factors underlying canine aggression. Here, we describe the characterization of three genes of the canine serotonergic system: the serotonin receptor 1A and 2A gene (htr1A and htr2A) and the serotonin transporter gene (slc6A4). We isolated canine bacterial artificial chromosome clones containing these genes and designed oligonucleotides for genomic sequencing of coding regions and intron-exon boundaries. Golden retrievers were analyzed for DNA sequence variations. We found two nonsynonymous single nucleotide polymorphisms (SNPs) in the coding sequence of htr1A; one SNP close to a splice site in htr2A; and two SNPs in slc6A4, one in the coding sequence and one close to a splice site. In addition, we identified a polymorphic microsatellite marker for each gene. Htr1A is a strong candidate for involvement in the domestication of the dog. We genotyped the htr1A SNPs in 41 dogs of seven breeds with diverse behavioral characteristics. At least three SNP haplotypes were found. Our results do not support involvement of the gene in domestication.

Transmembrane Signaling in the Brain by Serotonin, A Key Regulator of Physiology and Emotion

Serotonin (5-HT) is an ancient chemical that plays a crucial functional role in almost every living organism. It regulates platelet aggregation, activation of immune cells, and contraction of stomach and intestinal muscles. In addition, serotonin acts as a neurotransmitter in the brain and the peripheral nervous system. These activities are initiated by the binding of serotonin to 15 or more receptors that are pharmacologically classified into seven groups, 5-HT1 through 5-HT7. Each group is further divided into subgroups of receptors that are homologous but are encoded by discrete genes. With the exception of the 5-HT3 receptor--a cation channel--all of the others are G protein-coupled receptors that potentially activate or inhibit a large number of biochemical cascades. This review will endeavor to compare and contrast such signaling pathways with special attention to their tissue-specific occurrence, their possible role in immediate effects on covalent modification of other proteins, and relatively slower effects on gene expression, physiology and behavior.

Metabolism and Receptor Binding of Serotonin in Brain Structures During Performance of a Conditioned Passive Avoidance Response

The levels of serotonin and its metabolite 5-hydroxyindoleacetic acid, monoamine oxidase activity, and the specific binding of the radioligand [3H]serotonin were measured in the prefrontal cortex, striatum, amygdaloid complex, hippocampus, and periacqueductal gray matter of the midbrain in rats at different time points after training to a conditioned passive avoidance reaction. Changes in serotoninergic activity were found to be characteristic only for the process of reproducing the conditioned reaction. The metabolism and serotonin receptor binding in these brain structures did not change immediately after the training period or one day after this, or in conditions of failure to reproduce the reaction because of amnesia, or in untrained animals. The involvement of the brain serotoninergic system in the process of performing the conditioned reaction was found to demonstrate a spatial-structural selectivity: the metabolism and receptor binding of serotonin changed in the amygdaloid complex, periacqueductal gray matter, and the striatum, while no changes were seen in the hippocampus or prefrontal cortex. All three brain structures showed decreases in [3H]serotonin receptor binding of. Serotonin levels did not change, though the amygdaloid complex and periacqueductal gray matter showed increases in oxidative deamination of serotonin and increases in the active transport of the metabolite, while the striatum showed decreases in serotonin catabolism. The differences in the catabolism of this neurotransmitter suggest that the decrease in serotonin receptor binding in these brain structures depends on different synaptic processes--presynaptic in the striatum and postsynaptic in the amygdaloid complex and periacqueductal gray matter. It is concluded that the decrease in the functional activity of serotoninergic transmission in the amygdaloid complex and periacqueductal gray matter is one of the mechanisms involved in activation of the emotiogenic system triggering the process of reproduction of the memory trace.