miRNA-203b-3p Induces Acute and Chronic Pruritus through 5-HTR2B and TRPV4.

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch through transient receptor potential channels signaling are poorly understood. In this study, we show that genetic deletion or pharmacological antagonism of TRPV4 attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several microRNAs, including the miR-203b-3p, which induced a calcium ion response in rodent dorsal root ganglion neurons and scratching behavior in mice through 5-HTR2B activation and the protein kinase C‒dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular microRNA that can behave as an itch promoter through 5-HTR2B and TRPV4.

Fingerprint-Based Machine Learning Approach to Identify Potent and Selective 5-HT2BR Ligands.

The identification of subtype-selective GPCR (G-protein coupled receptor) ligands is a challenging task. In this study, we developed a computational protocol to find compounds with 5-HT2BR versus 5-HT1BR selectivity. Our approach employs the hierarchical combination of machine learning methods, docking, and multiple scoring methods. First, we applied machine learning tools to filter a large database of druglike compounds by the new Neighbouring Substructures Fingerprint (NSFP). This two-dimensional fingerprint contains information on the connectivity of the substructural features of a compound. Preselected subsets of the database were then subjected to docking calculations. The main indicators of compounds' selectivity were their different interactions with the secondary binding pockets of both target proteins, while binding modes within the orthosteric binding pocket were preserved. The combined methodology of ligand-based and structure-based methods was validated prospectively, resulting in the identification of hits with nanomolar affinity and ten-fold to ten thousand-fold selectivities.

5-Hydroxytryptamine Changes under Different Pretreatments on Rat Models of Myocardial Infarction and/or Depression.

BACKGROUND:: Psychocardiological researches have suggested a central role of 5-hydroxytryptamine (5-HT) on psychocardiological mechanism. This study aimed to further explore the central role of 5-HT and pretreatment effects of XinLingWan on rats with myocardial infarction (MI) and/or depression. METHODS:: Ninety Sprague-Dawley rats were randomly divided into three groups: MI group, depression group, and MI + depression group (n = 30 in each group). Each group was then divided into three subgroups (n = 10 in each subgroup): a negative control subgroup (NCS), a Western medicine subgroup (WMS), and a traditional Chinese medicine subgroup (TCMS), which were received pretreatment once a day for 4 weeks by saline, 20 mg/kg sertraline mixed with 2 ml saline, and 40 mg/kg XingLingWan mixed with 2 ml saline, respectively. Different rat models were established after different pretreatments. Rats were then sacrificed for detection of serum 5-HT, platelet 5-HT, 5-HT2A receptors (5-HT2AR), and serotonin transporter (SERT). Data were analyzed by one-way analysis of variance (ANOVA) and least-significant difference (LSD) testing. RESULTS:: MI group: compared with NCS, there was a significant increase in WMS and TCMS of serum 5-HT (176.15 ± 11.32 pg/ml vs. 334.50 ± 29.09 pg/ml and 474.04 ± 10.86 pg/ml, respectively, both P = 0.000), platelet 5-HT (129.74 ± 27.17 pg/ml vs. 322.24 ± 11.60 pg/ml and 340.4 5 ± 17.99 pg/ml, respectively, both P = 0.000); depression group: compared with NCS, there was a significant increase in WMS and TCMS of serum 5-HT (194.69 ± 5.09 pg/ml vs. 326.21 ± 39.98 pg/ml and 456.33 ± 23.12 pg/ml, respectively, both P = 0.000), platelet 5-HT (175.15 ± 4.07 pg/ml vs. 204.56 ± 18.59 pg/ml and 252.03 ± 22.26 pg/ml, respectively, P = 0.004 and P = 0.000, respectively); MI + depression group: compared with NCS, there was a significant increase in both WMS and TCMS of serum 5-HT (182.50 ± 10.23 pg/ml vs. 372.55 ± 52.23 pg/ml and 441.76 ± 23.38 pg/ml, respectively, both P = 0.000) and platelet 5-HT (180.83 ± 11.08 pg/ml vs. 221.12 ± 22.23 pg/ml and 265.37 ± 29.49 pg/ml, respectively, P = 0.011 and P = 0.000, respectively). CONCLUSIONS:: By elevating the amount of 5-HT and modulating 5-HT2AR and SERT levels in serum and platelets, XinLingWan and sertraline were found to exert pretreatment effect on rat models of MI and/or depression.

Effects of Ginseng Fruit Saponins on Serotonin System in Sprague-Dawley Rats with Myocardial Infarction, Depression, and Myocardial Infarction Complicated with Depression.

BACKGROUND: Our previous studies have demonstrated that the levels of 5-hydroxytryptamine (5-HT) and 5-HT 2A receptor (5-HT2AR) in serum and platelet were associated with depression and myocardial infarction (MI), and pretreatment with ginseng fruit saponins (GFS) before MI and depression had an effect on the 5-HT system. In this study, the effects of GFS on the 5-HT system in the Sprague-Dawley (SD) rats with MI, depression, and MI + depression were evaluated. METHODS: A total of eighty SD rats were allocated to four groups: MI, depression, MI + depression, and control groups (n = 20 in each group). Each group included two subgroups (n = 10 in each subgroup): Saline treatment subgroup and GFS treatment subgroup. The levels of 5-HT, 5-HT2AR, and serotonin transporter (SERT) were quantified in serum, platelet lysate, and brain tissue through the enzyme-linked immunosorbent assay method, respectively. RESULTS: Compared with those in the saline treatment subgroups, the levels of 5-HT in serum and platelet lysate statistically significantly increased in the GFS treatment subgroups of MI, depression, and MI + depression groups (serum: all P = 0.000; platelet lysate: P = 0.002, 0.000, 0.000, respectively). However, the 5-HT levels in brain homogenate significantly decreased in the GFS treatment subgroups compared with those in the saline treatment subgroups in MI and depression groups (P = 0.025 and 0.044 respectively), and no significant difference was observed between saline and GFS treatment subgroups in MI + depression group (P = 0.663). Compared with that in GFS treatment subgroup of control group, the 5-HT2AR levels in the platelet lysate significantly decreased in GFS treatment subgroups of MI, depression, and MI + depression groups (all P = 0.000). Compared to those in the saline treatment subgroups, the serum SERT levels significantly decreased in the GFS treatment subgroups in MI, depression, and MI + depression groups (P = 0.009, 0.038, and P = 0.001, respectively), while the SERT levels of platelet lysate significantly decreased in GFS treatment subgroup of MI group (P = 0.000), significantly increased in GFS treatment subgroup of depression group (P = 0.019), and slightly changed in GFS treatment subgroup of MI + depression group (P = 0.219). No significant changes for SERT levels in brain homogenate could be found between the saline and GFS treatment subgroups in MI, depression, and MI + depression groups (P = 0.421, 0.076 and P = 0.642). CONCLUSIONS: This study indicated that GFS might inhibit the reuptake of 5-HT from serum to platelet according to decreased 5-HT2AR in platelet and SERT in serum and platelet. The change of 5-HT in serum after GFS treatment was inconsistent with that in the brain. It seemed that GFS could not pass through the blood-brain barrier to affect the central serotonergic system.

Impairment of gastric accommodation induced by water-avoidance stress is mediated by 5-HT2B receptors.

BACKGROUND: Psychological stress has been shown to impair gastric accommodation (GA), but its mechanism has not been elucidated. This study was conducted to clarify the role of 5-HT2B receptors in a guinea pig model of stress-induced impairment of GA. METHODS: Gastric accommodation was evaluated by measuring the intrabag pressure in the proximal stomach after administration of a liquid meal. The guinea pigs were subjected to water-avoidance stress. The role of 5-HT2B receptors in impairment of GA was investigated by administering a 5-HT2B receptor agonist (BW723C86) or antagonist (SB215505), the traditional Japanese medicine rikkunshito (RKT), a muscarinic M3 receptor antagonist (1,1-dimethyl-4-diphenylacetoxypiperidium iodide [4-DAMP]), or a nitric oxide synthase inhibitor (Nω -nitro-L-arginine [L-NNA]). KEY RESULTS: In normal animals, liquid meal-induced GA was inhibited by BW723C86, but was not affected by SB215505. The inhibition of GA by BW723C86 was reversed by co-administration of 4-DAMP. Compared to normal animals, GA in stressed animals was significantly inhibited. SB215505 and RKT significantly suppressed stress-induced impairment of GA. After meal administration, the level of cyclic guanosine monophosphate in gastric fundus tissue increased by approximately twofold in normal animals, but did not change in stressed animals. The inhibition of GA by L-NNA was suppressed by SB215505 or RKT. At a dose that did not affect GA in normal animals, BW723C86 exacerbated the impairment of GA in stressed animals. CONCLUSIONS AND INFERENCES: Stress-induced impairment of GA may be mediated by an increased responsiveness of 5-HT2B receptors, and activation of the 5-HT2B receptor signaling pathway may have an inhibitory effect on nitric oxide function.

Contribution of serotonin to cardiac remodeling associated with hypertensive diastolic ventricular dysfunction in rats.

OBJECTIVE: Left-ventricular hypertrophy and interstitial fibrosis are the main pathophysiological factors of heart failure with preserved ejection fraction. Blockade of the serotonin 5-HT2B receptor (5-HT2BR) has been shown to reduce cardiac hypertrophy, oxidative stress, and extracellular cell matrix activation. In this study, we evaluated the effects of the 5-HT2BR blockade, on hemodynamic and cardiac remodeling, in spontaneously hypertensive rats (SHRs) that display a diastolic dysfunction with preserved ejection fraction. METHOD: Thirty-seven-week-old SHRs were randomized in four groups receiving either saline, the selective 5-HT2BR antagonist RS-127445 (1 mg/kg per day), a calcium channel blocker nicardipine (6 mg/kg per day), or RS-127445 + nicardipine. During the 14 weeks of treatment period, cardiac function and blood pressure were monitored by echocardiography and tail-cuff. Finally, electrocardiograms and invasive hemodynamics were obtained before blood collection. Heart was analyzed for morphology and mRNA expression. A complementary study evaluated the cardiac and vascular effects of serotonin on wild-type and mice knockout for the 5-HT2BR (Htr2B) and/or the 5-HT2AR (Htr2A). RESULTS: Despite the left ventricular 5-HT2BR overexpression, 5-HT2BR blockade by RS-127445 did not affect left ventricular hypertrophy and fibrosis in SHRs. This antagonist did not improve diastolic dysfunction, neither alone nor in combination with nicardipine, although it induced plasma brain natriuretic peptide decrease. Moreover, RS-127445 amplified subendocardial fibrosis and favored left ventricular dilatation. Finally, a subendocardial left ventricular fibrosis was induced by chronic serotonin in wild-type mice, which was increased in Htr2B animals, but prevented in Htr2A and Htr2A/2B mice, and could be explained by a contribution of the endothelial 5-HT2BRs to coronary vasodilatation. CONCLUSION: This work is the first to identify a cardioprotective function of the 5-HT2BR in an integrated model of diastolic dysfunction with preserved ejection fraction.

Synthesis and pharmacological evaluation of N-benzyl substituted 4-bromo-2,5-dimethoxyphenethylamines as 5-HT2A/2C partial agonists.

N-Benzyl substitution of phenethylamine 5-HT2A receptor agonists has dramatic effects on binding affinity, receptor selectivity and agonist activity. In this paper we examine how affinity for the 5-HT2A/2C receptors are influenced by N-benzyl substitution of 4-bromo-2,5-dimethoxyphenethylamine derivatives. Special attention is given to the 2' and 3'-position of the N-benzyl as such compounds are known to be very potent. We found that substitutions in these positions are generally well tolerated. The 2'-position was further examined using a range of substituents to probe the hydrogen bonding requirements for optimal affinity and selectivity, and it was found that small changes in the ligands in this area had a profound effect on their affinities. Furthermore, two ligands that lack a 2'-benzyl substituent were also found to have high affinity contradicting previous held notions. Several high-affinity ligands were identified and assayed for functional activity at the 5-HT2A and 5-HT2C receptor, and they were generally found to be less efficacious agonists than previously reported N-benzyl phenethylamines.

Effect of immobilization stress on the appetite and stomach ghrelin expression in maternal mice.

Maternal stress exerts long-lasting postnatal growth on offspring, which persist into adulthood. However, the effect of maternal stress on appetizing system has not been widely reported. In this study, we found that maternal immobilization stress (IS) during lactation resulted in low body weight and food intake. Immunohistochemistry showed an increase in stomach ghrelin protein expression. The central regulation of body weight and food intake occurs in the hypothalamus, which contains multiple neuronal systems that play important roles in the regulation of energy homeostasis. These systems including multiple neuropeptides involve in the ghrelin pathway of appetite regulation. Therefore, real time reverse transcription polymerase chain reaction (RT-PCR) was used to measure the change of mRNA expression of ghrelin pathway related hormones in order to explore the mechanisms involved in the appetite regulation. Expression levels of the hypothalamic 5-hydroxytryptamine 2c receptor (5-HT2cR) and 5-HT2bR, which are essential for the development and function of ghrelin and leptin, were decreased, as well as those of corticotrophin releasing factor (CRF) and pro-opiomelanocortin (POMC). While the expression of growth hormone secretagogue receptor (GHSR), neuropeptide-Y (NPY) and agouti-related protein (AgRP) showed an increase with significant difference. These results suggest that stress in a postpartum mother has persistent effects on the body weight of their offspring. Increased ghrelin and decreased leptin expression in the stomach may play a role in these effects.

Safety Pharmacology assessment of drugs with biased 5-HT(2B) receptor agonism mediating cardiac valvulopathy.

INTRODUCTION: The rhythmic opening and tightly closing of cardiac valve leaflets are cardiac cyclic events imposing to blood a unidirectional course along the vascular tree. Drugs with 5-HT2B agonism properties can seriously compromise this biological function critical for hemodynamic efficiency as their intrinsic pro-fibrotic effects can, with time, make valvular coaptation blood regurgitant. TOPICS COVERED: Cardiac valve anatomy, physiology and pathology as well as 5-HT2B receptor properties (coupling, effects mediated, biased agonism) are briefly exposed. Approaches to unveil 5-HT2B receptor liability of drug candidates are detailed. In silico computational models can rapidly probe molecules for chemical signatures associated with 5-HT2B receptor affinity. In vitro radioligand competition assays allow quantifying receptor binding capacity (Ki, IC50), the pharmacological nature (agonism, antagonism) of which can be ascertained from cytosolic second messenger (inositol phosphates, Ca(++), MAPK2) changes. Potencies calculated from the latter data may exhibit variability as they are dependent upon the readout measured and the experimental conditions (e.g., receptor density level of cell material expressing human 5-HT2B receptors). The in vivo valvulopathy effects of 5-HT2B receptor agonists can be assessed by echocardiographic measurements and valve histology in rats chronically treated with the candidate drug. Finally, safety margins derived from from nonclinical and clinical data are evaluated in terms of the readout, usefulness and scientific reliability. DISCUSSION: The Safety Pharmacology toolbox for detecting possible 5-HT2B receptor agonism liabilities of candidate drugs requires meticulous optimization and validation of all its (in silico, in vitro and in vivo) components to perfect its human predictability power. In particular, since 5-HT2B receptor agonism is biased in nature, the most predictive readout(s) of valvular liability should be identified and prioritized in keeping with best scientific practice teachings.

Rikkunshito, a Japanese kampo medicine, ameliorates decreased feeding behavior via ghrelin and serotonin 2B receptor signaling in a novelty stress murine model.

We investigated the effects of rikkunshito (RKT), a ghrelin signal enhancer, on the decrease in food intake after exposure to novelty stress in mice. RKT administration (500 mg/kg, per os) improved the decrease in 6 h cumulative food intake. In control mice, the plasma acylated ghrelin levels significantly increased by 24 h fasting. In contrast, the acylated ghrelin levels did not increase by fasting in mice exposed to the novelty stress. RKT administration to the novelty stress mice showed a significant increase in the acylated ghrelin levels compared with that in the distilled-water-treated control mice. Food intake after administering serotonin 2B (5-HT(2B)) receptor antagonists was evaluated to clarify the role of 5-HT(2B) receptor activation in the decrease in feeding behavior after novelty stress. SB215505 and SB204741, 5-HT(2B) receptor antagonists, significantly improved the decrease in food intake after exposure to novelty stress. A component of RKT, isoliquiritigenin, prevented the decrease in 6 h cumulative food intake. Isoliquiritigenin showed 5-HT(2B) receptor antagonistic activity in vitro. In conclusion, the results suggested that RKT improves the decrease in food intake after novelty stress probably via 5-HT(2B) receptor antagonism of isoliquiritigenin contained in RKT.

Predictive in silico studies of human 5-hydroxytryptamine receptor subtype 2B (5-HT2B) and valvular heart disease.

Serotonin (5-hydroxytryptamine, 5-HT) receptors are neuromodulator neurotransmitter receptors which when activated trigger a signal transduction cascade within cells resulting in cell-cell communication. 5-hydroxytryptamine receptor 2B (5-HT2B) is a subtype of the seven members of 5-hydroxytrytamine receptors family which is the largest member of the super family of 7-transmembrane G-protein coupled receptors (GPCRs). Not only do 5-HT receptors play physiological roles in the cardiovascular system, gastrointestinal and endocrine function as well as the central nervous system, but they also play a role in behavioral functions. In particular 5-HT2B receptor is widely spread with regards to its distribution throughout bodily tissues and is expressed at high levels in the lungs, peripheral tissues, liver, kidneys and prostate, just to name a few. Hence 5-HT2B participates in multiple biological functions including CNS regulation, regulation of gastrointestinal motality, cardiovascular regulation and 5-HT transport system regulation. While 5-HT2B is a viable drug target and has therapeutic indications for treating obesity, psychosis, Parkinson's disease etc. there is a growing concern regarding adverse drug reactions, specifically valvulopathy associated with 5-HT2B agonists. Due to the sequence homology experienced by 5-HT2 subtypes there is also a concern regarding the off-target effects of 5-HT2A and 5-HT2C agonists. The concepts of sensitivity and subtype selectivity are of paramount importance and now can be tackled with the aid of in silico studies, especially cheminformatics, to develop models to predict valvulopathy associated toxicity of drug candidates prior to clinical trials. This review has highlighted three in silico approaches thus far that have been successful in either predicting 5-HT2B toxicity of molecules or identifying important interactions between 5-HT2B and drug molecules that bring about valvulopathy related toxicities.

Phosphorylation of Akt/GSK-3ß/eNOS amplifies 5-HT2B receptor blockade mediated anti-hypertrophic effect in rats.

Herein, we studied the cross talk between 5-HT(2B) receptor blocker (SB-204741) and GSK-3ß inhibitor (SB-216763) in isoproterenol-induced cardiac hypertrophy for 28 days. SB-204741 treatment significantly ameliorated (P<0.05) myocardial dysfunction, myocyte area, fibrosis and myocardial architecture in isoproterenol insulted myocardium. Moreover, this improvement in functional and morphological changes was associated with suppression of hypertrophic (BNP and CK-MB), inflammatory (IKK-ß/NF-κB/TNF-α and CRP), and apoptotic markers (TUNEL positivity and Bax expression) along with phosphorylation of Akt/GSK-3ß/ß-catenin/eNOS. Intriguingly, co-treatment with GSK-3ß inhibitor (P<0.01) further amplified the anti-hypertrophic effect of SB-204741 (P<0.05) such that the effect was indistinguishable from that of vehicle treated rats. Thus, 5-HT(2B) receptor blockade mediated anti-hypertrophic effect is atleast in part is governed through phosphorylation of Akt/GSK-3ß/ß-catenin/eNOS via attenuating inflammatory and apoptotic pathways.

Fluoxetine affects GluK2 editing, glutamate-evoked Ca(2+) influx and extracellular signal-regulated kinase phosphorylation in mouse astrocytes.

BACKGROUND: We sought to study the effects of chronic exposure to fluoxetine - a selective serotonin reuptake inhibitor (SSRI) and specific 5-HT(2B) receptor agonist in astrocytes - on the expression of kainate receptors (GluK1-5) in cultured astrocytes and in intact brains in mice and on GluK2 editing by adenosine deaminase acting on RNA (ADAR), as well as the ensuing effects of fluoxetine on glutamate-mediated Ca(2+) influx and extracellular signal-regulated kinase (ERK)(1/2) phosphorylation in astrocytes. METHODS: We performed reverse transcription-polymerase chain reaction (PCR) to assess mRNA expression. We analyzed RNA editing with amplification refractory mutation system PCR and complementary DNA sequencing. Protein expression and ERK phosphorylation were assessed using Western blots. We studied gene silencing with specific small interfering RNAs (siRNA), and we studied intracellular Ca(2+) using fluorometry. RESULTS: All GluK subunits were present in the brain in vivo, and GluK2-5 subunits were present in cultured astrocytes. Fluoxetine upregulated GluK2 and ADAR2. Enhanced GluK2 editing by fluoxetine abolished glutamate-mediated increases in intra cellular Ca(2+) and ERK(1/2) phosphorylation. Enhanced editing of GluK2 was prevented by siRNA against the 5-HT(2B) receptor or ADAR2. LIMITATIONS: Limitations of our study include the use of an in vitro system, but our cultured cells in many respects behave like in vivo astrocytes. CONCLUSION: Fluoxetine alters astrocytic glutamatergic function.

Drug-induced valvulopathy: an update.

Drug-induced valvulopathy is a serious liability for certain compound classes in development and for some marketed drugs intended for human use. Reports of valvulopathy led to the withdrawal of fenfluramines (anorexigens) and pergolide (antiparkinson drug) from the United States market in 1997 and 2007, respectively. The mechanism responsible for the pathogenesis of valvulopathy by these drugs is likely a result of an "off-target" effect via activation of 5-hydroxytryptamine (5-HT) 2B receptor (5-HT2BR) expressed on heart valve leaflets. Microscopically, the affected valve leaflets showed plaques of proliferative myofibroblasts in an abundant extracellular matrix, composed primarily of glycosaminoglycans. However, the valvular effects caused by fenfluramines and pergolide were not initially predicted from routine preclinical toxicity studies, and to date there are no specific validated animal models or preclinical/toxicologic screens to accurately predict drug-induced valvulopathy. This review covers the structure and function of heart valves and highlights major advances toward understanding the 5-HT2BR-mediated pathogenesis of the lesion and subsequently, development of appropriate animal models using novel techniques/experiments, use of functional screens against 5-HT2BR, and more consistent sampling and pathologic evaluation of valves in preclinical studies that will aid in avoidance of future drug-induced valvulopathy in humans.

Increased expression of 5-hydroxytryptamine2A/B receptors in idiopathic pulmonary fibrosis: a rationale for therapeutic intervention.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited responsiveness to available treatments. It is characterised by epithelial cell injury, fibroblast activation and proliferation and extracellular matrix deposition. Serotonin (5-hydroxytryptamine; 5-HT) induces fibroblast proliferation via the 5-HTR(2A) and 5-HTR(2B) receptors, but its pathophysiological role in IPF remains unclear. A study was undertaken to determine the expression of 5-HT receptors in IPF and experimental lung fibrosis and to investigate the effects of therapeutic inhibition of 5-HTR(2A/B) signalling on lung fibrosis in vivo and in vitro. METHODS AND RESULTS: Quantitative RT-PCR showed that the expression of 5-HTR(1A/B) and 5-HTR(2B) was significantly increased in the lungs of patients with IPF (n=12) and in those with non-specific interstitial pneumonia (NSIP, n=6) compared with transplant donors (n=12). The expression of 5-HTR(2A) was increased specifically in IPF lungs but not in NSIP lungs. While 5-HTR(2A) protein largely localised to fibroblasts, 5-HTR(2B) localised to the epithelium. To assess the effects of 5HTR(2A/B) inhibition on fibrogenesis in vivo, mice were subjected to bleomycin-induced lung fibrosis and treated with the 5-HTR(2A/B) antagonist terguride (or vehicle) in a therapeutic approach (days 14-28 after bleomycin). Terguride-treated mice had significantly improved lung function and histology and decreased collagen content compared with vehicle-treated mice. Functional in vitro studies showed that terguride is a potent inhibitor of transforming growth factor ß(1)- or WNT3a-induced collagen production. CONCLUSION: The studies revealed an increased expression of 5-HTR(2A) specifically in IPF. Blockade of 5-HTR(2A/B) signalling by terguride reversed lung fibrosis and is thus a promising therapeutic approach for IPF.

Characterization of the 5-HT2b receptor in evaluation of aequorin detection of calcium mobilization for miniaturized GPCR high-throughput screening.

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.

Rikkunshito, an herbal medicine, suppresses cisplatin-induced anorexia in rats via 5-HT2 receptor antagonism.

BACKGROUND & AIMS: Chemotherapy with an anticancer agent generally causes gastrointestinal tract disorders such as vomiting and anorexia, but the mechanism remains unclear. Rikkunshito, a kampo preparation, is known to alleviate such adverse reactions. In this study, we attempted to clarify the mechanism. METHODS: We investigated the decreases of plasma acylated-ghrelin level and food intake caused by cisplatin, serotonin (5-HT), 5-HT agonists, and vagotomy as well as the decrease-suppressing effects of rikkunshito and 5-HT antagonists. In addition, binding affinities of rikkunshito components were determined in receptor-binding assays using 5-HT2B and 5-HT2C receptors. RESULTS: Cisplatin, 5-HT, BW723C86 (5-HT2B-receptor agonist), and m-chlorophenylpiperazine HCl (5-HT2C agonist) markedly decreased plasma acylated-ghrelin levels, although 5-HT3 and 5-HT4 agonists had no effect. In contrast, 5-HT2B and 5-HT2C antagonists suppressed the cisplatin-induced decrease of plasma acylated-ghrelin level and food intake. Administration of rat ghrelin improved the cisplatin-induced decrease in food intake. Vagotomy decreased the plasma acylated-ghrelin level, which was decreased further by cisplatin. Rikkunshito suppressed such cisplatin-induced decreases of plasma acylated-ghrelin level and food intake. The suppressive effect of rikkunshito was blocked by a ghrelin antagonist. Components of rikkunshito, 3,3',4',5,6,7,8-heptamethoxyflavone, hesperidin, and iso-liquiritigenin showed a 5-HT2B-antagonistic effect in vitro, and oral administration of rikkunshito suppressed the cisplatin-induced decrease in the plasma acylated-ghrelin level. CONCLUSIONS: The cisplatin-induced decreases of the plasma acylated-ghrelin level and food intake are mediated by 5-HT2B/2C receptors and suppressed by flavonoids in rikkunshito.

A comprehensive in vitro screening of d-, l-, and dl-threo-methylphenidate: an exploratory study.

dl-Methylphenidate (MPH) has been widely used to treat attention-deficit/hyperactivity disorder (ADHD) for the last half century. It had been exclusively available in the racemic form, i.e., a 50:50 mixture of d- and l-isomers. However, a single enantiomer formulation, d-MPH (dexmethylphenidate), became available for general clinical use in 2002. For this reason, the intrinsic pharmacological differences in the effects of d- and l-MPH have recently come under intense investigation. The primary therapeutic effects of MPH are generally recognized to reside in the d-isomer. The present investigation provides quantitative values for a broad range of receptor-level interactions of the individual MPH isomers to better characterize the distinction between dl-MPH versus d-MPH versus l-MPH as it relates to binding affinity at sites associated with relevant central nervous system (CNS) pharmacology, as well as peripheral physiology. Overall, there were few differences in binding affinities between d-MPH and the racemate whereas there were more apparent differences between d-MPH and l-MPH. d-MPH exhibited prominent affinity at the norepinephrine transporter (NET) site, even exceeding such affinity at the dopamine transporter (DAT). These results further demonstrate that affinity for catecholaminergic sites largely resides in the d-MPH isomer. Although binding affinity was not demonstrable at the serotonin (5-HT) transporter site (SERT), novel findings of the study included affinity for the 5-HT1A and 5-HT2B receptor sites for both d- and l-MPH, with d-MPH exerting by far the most predominant effects at these sites. Thus, the emerging data of favorable therapeutic effects of ADHD treatment with d-MPH (and dl-MPH) may be underpinned by affinity and potential pharmacologic effects at NET and DAT sites, as well as sites relevant to serotonergic neurotransmission that may modulate mood, cognition, and motor behavior. However, the present exploratory studies reflect receptor binding affinities only. The specific pharmacological activities (i.e., agonism vs. antagonism) of these compounds await further exploration.

Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes.

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.

Optical coding of mammalian cells using semiconductor quantum dots.

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.