Activation Of 5-HT1B Receptors Research Articles

Preclinical mitigation of 5-HT2B agonism-related cardiac valvulopathy revisited

Cardiac valvulopathy (Cardiac Valve Disease; CVD) associated with off-target activation of the 5-hydroxytryptamine (5-HT) 2B receptor has been well recognized, but is still poorly predicted during drug development. The regulatory guidance proposes the use of 5-HT2B binding data (i.e., Ki values) and free maximum therapeutic exposure (Cmax) to calculate safety margins as a threshold of detection (>10) for eliminating the risk of drug-induced cardiac valvulopathy. In this paper, we provide additional recommendations for preclinical prediction of CVD risk based on clinical pharmacodynamic and pharmacokinetic data obtained from drugs with or without 5-HT2B receptor activation. Our investigations showed that 5-HT2B agonist affinity of molecules tested in an in vitro 5-HT2B cell-based functional assay, placed in perspective to their sustained plasma exposure (AUCs) and not to their peak plasma exposure, Cmax (i.e., maximum therapeutic exposure) provide a solid basis for interpreting 5-HT2B data, for calculating safety margins and then, accurately differentiate drugs associated with a clinical risk of CVD from those which are not (despite having some agonist 5-HT2B activity). In addition, we discuss the risk of multi-organ fibrosis linked to 5-HT2B receptor activation, often underestimated, however well reported in FAERS for 5-HT2B agonists. We believe that our recommendations have the potential to mitigate the risk for the clinical development of CVD and fibrosis.

Molecular and cellular basis of praziquantel action in the cardiovascular system.

The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by the activation of host serotonergic 5-HT2B receptors (5-HT2BRs). However, the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall have not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F3406.51 and F3416.52 (transmembrane helix 6, TM6) as well as L209EL2 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.

Cardiovascular safety of new drugs for the acute and preventive treatment of migraine: gepants and ditans

Migraine is a prevalent neurological condition characterised by disabling headache attacks. In recent decades, new drugs have been developed specifically for the acute and preventive treatment of migraine based on its pathophysiology. These include calcitonin gene-related peptide (CGRP) antagonists (CGRP) (gepants) and selective serotoninergic 5-HT1F receptor agonists (ditans). CGRP is a neuropeptide released by trigeminal terminals that acts as a vasodilator, causes neurogenic inflammation and thus generates pain and sensitisation in migraine. It also has a powerful vasodilatory action and is involved in cardiovascular regulation, which is why numerous studies are under way to assess the vascular safety of acting against CGRP. The high selectivity of ditans for the serotoninergic 5-HT1F receptor with a low affinity for other serotoninergic receptors seems to translate into little or no vasoconstrictor effect, which is mediated by the activation of 5-HT1B receptors. The aim of our study is to review the cardiovascular safety demonstrated by these new drugs for the treatment of migraine by analysing the evidence published to date. We conducted a literature search in the PubMed database and a review of clinical trials published at clinicaltrial.gov. We included literature reviews, meta-analyses and clinical trials in English and Spanish. We analysed reported cardiovascular adverse effects. Based on the results published to date, we can conclude that the cardiovascular safety profile of these new treatments is favourable. Longer-term safety studies are needed to confirm these results.

Normal Pregnancy-Induced Islet Beta Cell Proliferation in Mouse Models That Are Deficient in Serotonin-Signaling.

During mouse pregnancy placental lactogens stimulate prolactin receptors on pancreatic islet beta cells to induce expression of the tryptophan hydroxylase Tph1, resulting in the synthesis and secretion of serotonin. Presently, the functional relevance of this phenomenon is unclear. One hypothesis is that serotonin-induced activation of 5-HT2B receptors on beta cells stimulates beta cell proliferation during pregnancy. We tested this hypothesis via three different mouse models: (i) total Tph1KO mice, (ii) 129P2/OlaHsd mice, which are incompetent to upregulate islet Tph1 during pregnancy, whereas Tph1 is normally expressed in the intestine, mammary glands, and placenta, and (iii) Htr2b-deficient mice. We observed normal pregnancy-induced levels of beta cell proliferation in total Tph1KO mice, 129P2/OlaHsd mice, and in Htr2b-/- mice. The three studied mouse models indicate that islet serotonin production and its signaling via 5-HT2B receptors are not required for the wave of beta cell proliferation that occurs during normal mouse pregnancy.

Serotonin 5-HT1A and 5-HT1B receptor-mediated inhibition of glutamatergic transmission onto rat basal forebrain cholinergic neurones.

Cholinergic neurones in the basal forebrain (BF) project into various brain regions and receive excitatory inputs from the cortex and brain stem. These cholinergic neurones receive serotonergic fibres from the dorsal raphe nuclei. This study was aimed to elucidate serotonin (5-HT)-induced modulation of glutamatergic transmission onto rat BF cholinergic neurones identified with Cy3-192IgG. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation. Bath application of either 5-HT, the 5-HT1A receptor agonist 8-OH-DPAT (DPAT), or the 5-HT1B receptor agonist CP93129 (CP), inhibited the amplitude of EPSCs. In the presence of both 5-HT1A and 5-HT1B receptor antagonists, the 5-HT-induced effect disappeared. The paired-pulse ratio (PPR) and coefficient of variation (CV) of the EPSCs were increased by CP, whereas DPAT had no effect on PPR or CV. DPAT inhibited the inward currents induced by puff application of l-glutamate, which were unaffected by CP. DPAT suppressed the amplitude of miniature EPSCs (mEPSCs) without affecting their frequency. CP decreased the frequency of mEPSCs in more than half of the neurones examined, whereas the amplitude was unaffected. DPAT or CP alone inhibited the NMDA receptor-mediated currents. 5-HT-induced inhibition of EPSCs was reduced in the presence of ω-agatoxin TK (Aga). Furthermore, CP-induced inhibition of EPSCs was eliminated in the presence of Aga. DPAT-induced inhibition of EPSCs was unchanged in the presence of Aga. These results suggest that activation of 5-HT1A receptors reduces the sensitivity of postsynaptic glutamate receptors to glutamate, whereas presynaptic activation of 5-HT1B receptors inhibits glutamate release by blocking P/Q-type calcium channels. KEY POINTS: We performed a patch-clamp study to investigate serotonin (5-HT)-induced modulation of glutamatergic transmission onto cholinergic neurones in the rat basal forebrain slices. Excitatory postsynaptic currents (EPSCs) were inhibited by 5-HT as well as agonists of 5-HT1A or 5-HT1B receptors. 5-HT-induced inhibition was antagonized by co-application of 5-HT1A and 5-HT1B receptor antagonists. The effects of 5-HT receptor agonists on the paired-pulse ratio, coefficient of variation of EPSCs, inward currents induced by puff application of l-glutamate as well as miniature EPSCs suggest that activation of 5-HT1A receptors decreases the sensitivity of postsynaptic glutamate receptors to glutamate, whereas 5-HT1B receptors presynaptically inhibit glutamate release. The 5-HT1B agonist-induced inhibition was eliminated in the presence of a P/Q-type calcium channel blocker, whereas the 5-HT1A agonist still inhibited the EPSCs even in the presence of the blocker. The present study reveals different pre- and postsynaptic mechanisms underlying 5-HT1A and 5-HT1B receptor-mediated modulation of excitatory transmission.

Acute serotonin 1B/1A receptor activation impairs behavioral flexibility in C57BL/6J mice

Pharmacological activation of the serotonin (5-HT) 1B and 5-HT1A receptors has been shown to induce OCD-like perseverative circling and locomotor stereotypy in rodents. Although, several studies have examined how activation of these receptors facilitates these motor-associated OCD-like behaviors, it is not known how acute 5-HT1B and 5-HT1A activation impacts behavioral inflexibility, a common trait related to OCD. The current study examined how acute 5-HT1B/1A receptor agonist RU24969 treatment at 0.01, 0.1, and 1.0 mg/kg impacted behavioral flexibility in both female and male C57BL/6J mice. Behavioral flexibility was tested using a spatial reversal learning task, with probabilistic reward contingencies. In addition, locomotor activity and anxiety-like behaviors were also measured. RU24969 at 0.1 and 1.0 mg/kg impaired behavioral flexibility in both female and male C57BL/6J mice. RU24969 treatment at 1.0 mg/kg reduced locomotor activity in male mice, although RU24969 treatment did not significantly reduce locomotor activity in female mice. In the open field, 1.0 mg/kg elevated anxiety-like behavior in male mice only. Overall, these results demonstrate that acute 5-HT1B and 5-HT1A receptor activation leads to impairments in behavioral flexibility, a common trait associated with OCD.

Receptor Interaction Profiles of 4-Alkoxy-3,5-Dimethoxy-Phenethylamines (Mescaline Derivatives) and Related Amphetamines.

3,4,5-Trimethoxyphenethylamine (mescaline) is a psychedelic alkaloid found in peyote cactus. Related 4-alkoxy-3,5-dimethoxy-substituted phenethylamines (scalines) and amphetamines (3C-scalines) are reported to induce similarly potent psychedelic effects and are therefore potential novel therapeutics for psychedelic-assisted therapy. Herein, several pharmacologically uninvestigated scalines and 3C-scalines were examined at key monoamine targets in vitro. Binding affinity at human serotonergic 5-HT1A, 5-HT2A, and 5-HT2C, adrenergic α1A and α2A, and dopaminergic D2 receptors, rat and mouse trace amine-associated receptor 1 (TAAR1), and human monoamine transporters were assessed using target specific transfected cells. Furthermore, activation of human 5-HT2A and 5-HT2B receptors, and TAAR1 was examined. Generally, scalines and 3C-scalines bound with weak to moderately high affinity to the 5-HT2A receptor (K i = 150–12,000 nM). 3C-scalines showed a marginal preference for the 5-HT2A vs the 5-HT2C and 5-HT1A receptors whereas no preference was observed for the scalines. Extending the 4-alkoxy substituent increased 5-HT2A and 5-HT2C receptors binding affinities, and enhanced activation potency and efficacy at the 5-HT2A but not at the 5-HT2B receptor. Introduction of fluorinated 4-alkoxy substituents generally increased 5-HT2A and 5-HT2C receptors binding affinities and increased the activation potency and efficacy at the 5-HT2A and 5-HT2B receptors. Overall, no potent affinity was observed at non-serotonergic targets. As observed for other psychedelics, scalines and 3C-scalines interacted with the 5-HT2A and 5-HT2C receptors and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline.

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals.

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

Stress decreases serotonin tone in the nucleus accumbens in male mice to promote aversion and potentiate cocaine preference via decreased stimulation of 5-HT1B receptors.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

A Novel 5-HT1B Receptor Agonist of Herbal Compounds and One of the Therapeutic Uses for Alzheimer's Disease.

The serotonin receptor 5-HT1B is widely expressed in the central nervous system and has been considered a drug target in a variety of cognitive and psychiatric disorders. The anti-inflammatory effects of 5-HT1B agonists may present a promising approach for Alzheimer’s disease (AD) treatment. Herbal antidepressants used in the treatment of AD have shown functional overlap between the active compounds and 5-HT1B receptor stimulation. Therefore, compounds in these medicinal plants that target and stimulate 5-HT1B deserve careful study. Molecular docking, drug affinity responsive target stability, cellular thermal shift assay, fluorescence resonance energy transfer (FRET), and extracellular regulated protein kinases (ERK) 1/2 phosphorylation tests were used to identify emodin-8-O-β-d-glucopyranoside (EG), a compound from Chinese medicinal plants with cognitive deficit attenuating and antidepressant effects, as an agonist of 5-HT1B. EG selectively targeted 5-HT1B and activated the 5-HT1B-induced signaling pathway. The activated 5-HT1B pathway suppressed tumor necrosis factor (TNF)-α levels, thereby protecting neural cells against beta-amyloid (Aβ)-induced death. Moreover, the agonist activity of EG towards 5-HT1B receptor, in FRET and ERK1/2 phosphorylation, was antagonized by SB 224289, a 5-HT1B antagonist. In addition, EG relieved AD symptoms in transgenic worm models. These results suggested that 5-HT1B receptor activation by EG positively affected Aβ-related inflammatory process regulation and neural death resistance, which were reversed by antagonist SB 224289. The active compounds such as EG might act as potential therapeutic agents through targeting and stimulating 5-HT1B receptor for AD and other serotonin-related disorders. This study describes methods for identification of 5-HT1B agonists from herbal compounds and for evaluating agonists with biological functions, providing preliminary information on medicinal herbal pharmacology.

The Effect of Serotonin Receptor 5-HT1B on Lateral Inhibition between Spiny Projection Neurons in the Mouse Striatum.

The principal neurons of the striatum, the spiny projection neurons (SPNs), make inhibitory synaptic connections with each other via collaterals of their main axon, forming a local lateral inhibition network. Serotonin, acting via the 5-HT1B receptor, modulates neurotransmitter release from SPN terminals in striatal output nuclei, but the role of 5-HT1B receptors in lateral inhibition among SPNs in the striatum is unknown. Here, we report the effects of 5-HT1B receptor activation on lateral inhibition in the mouse striatum. Whole-cell recordings were made from SPNs in acute brain slices of either sex, while optogenetically activating presynaptic SPNs or fast-spiking interneurons (FSIs). Activation of 5-HT1B receptors significantly reduced the amplitude of IPSCs evoked by optical stimulation of both direct and indirect pathway SPNs. This reduction was blocked by application of a 5-HT1B receptor antagonist. Activation of 5-HT1B receptors did not reduce the amplitude of IPSCs evoked from FSIs. These results suggest a new role for serotonin as a modulator of lateral inhibition among striatal SPNs. The 5-HT1B receptor may, therefore, be a suitable target for future behavioral experiments investigating the currently unknown role of lateral inhibition in the function of the striatum.SIGNIFICANCE STATEMENT We show that stimulation of serotonin receptors reduces the efficacy of lateral inhibition between spiny projection neurons (SPNs), one of the biggest GABAergic sources in the striatum, by activation of the serotonin 5-HT1B receptor. The striatum receives serotonergic input from the dorsal raphe nuclei and is important in behavioral brain functions like learning and action selection. Our findings suggest a new role for serotonin in modulating the dynamics of neural interactions in the striatum, which extends current knowledge of the mechanisms of the behavioral effects of serotonin.

Systemic enhancement of serotonin signaling reverses social deficits in multiple mouse models for ASD.

Autism spectrum disorder (ASD) is a common set of heterogeneous neurodevelopmental disorders resulting from a variety of genetic and environmental risk factors. A core feature of ASD is impairment in prosocial interactions. Current treatment options for individuals diagnosed with ASD are limited, with no current FDA-approved medications that effectively treat its core symptoms. We recently demonstrated that enhanced serotonin (5-HT) activity in the nucleus accumbens (NAc), via optogenetic activation of 5-HTergic inputs or direct infusion of a specific 5-HT1b receptor agonist, reverses social deficits in a genetic mouse model for ASD based on 16p11.2 copy number variation. Furthermore, the recreational drug MDMA, which is currently being evaluated in clinical trials, promotes sociability in mice due to its 5-HT releasing properties in the NAc. Here, we systematically evaluated the ability of MDMA and a selective 5-HT1b receptor agonist to rescue sociability deficits in multiple different mouse models for ASD. We find that MDMA administration enhances sociability in control mice and reverses sociability deficits in all four ASD mouse models examined, whereas administration of a 5-HT1b receptor agonist selectively rescued the sociability deficits in all six mouse models for ASD. These preclinical findings suggest that pharmacological enhancement of 5-HT release or direct 5-HT1b receptor activation may be therapeutically efficacious in ameliorating some of the core sociability deficits present across etiologically distinct presentations of ASD.

Colonic Motility Is Improved by the Activation of 5-HT2B Receptors on Interstitial Cells of Cajal in Diabetic Mice

Constipation is commonly associated with diabetes. Serotonin (5-HT), produced predominantly by enterochromaffin (EC) cells via tryptophan hydroxylase 1 (TPH1), is a key modulator of gastrointestinal (GI) motility. However, the role of serotonergic signaling in constipation associated with diabetes is unknown. We generated EC cell reporter Tph1-tdTom, EC cell-depleted Tph1-DTA, combined Tph1-tdTom-DTA, and interstitial cell of Cajal (ICC)-specific Kit-GCaMP6 mice. Male mice and surgically ovariectomized female mice were fed a high-fat high-sucrose diet to induce diabetes. The effect of serotonergic signaling on GI motility was studied by examining 5-HT receptor expression in the colon and invivo GI transit, colonic migrating motor complexes (CMMCs), and calcium imaging in mice treated with either a 5-HT2B receptor (HTR2B) antagonist or agonist. Colonic transit was delayed in males with diabetes, although colonic Tph1+ cell density and 5-HT levels were increased. Colonic transit was not further reduced in diabetic mice by EC cell depletion. The HTR2B protein, predominantly expressed by colonic ICCs, was markedly decreased in the colonic muscles of males and ovariectomized females with diabetes. Ca2+ activity in colonic ICCs was decreased in diabetic males. Treatment with an HTR2B antagonist impaired CMMCs and colonic motility in healthy males, whereas treatment with an HTR2B agonist improved CMMCs and colonic motility in males with diabetes. Colonic transit in ovariectomized females with diabetes was also improved significantly by the HTR2B agonist treatment. Impaired colonic motility in mice with diabetes was improved by enhancing HTR2B signaling. The HTR2B agonist may provide therapeutic benefits for constipation associated with diabetes.

Pathological Insight into 5-HT2B Receptor Activation in Fibrosing Interstitial Lung Diseases.

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)2 receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.

Role of peripheral 5-HT5A receptors in 5-HT-induced cardiac sympatho-inhibition in type 1 diabetic rats

5-HT inhibits cardiac sympathetic neurotransmission in normoglycaemic rats, via 5-HT1B, 5-HT1D and 5-HT5A receptor activation. Since type 1 diabetes impairs the cardiac sympathetic innervation leading to cardiopathies, this study aimed to investigate whether the serotonergic influence on cardiac noradrenergic control is altered in type 1 diabetic rats. Diabetes was induced in male Wistar rats by streptozotocin (50 mg/kg, i.p.). Four weeks later, the rats were anaesthetized, pithed and prepared for producing tachycardic responses by electrical preganglionic stimulation (C7-T1) of the cardioaccelerator sympathetic outflow or i.v. noradrenaline bolus injections. Immunohistochemistry was performed to study 5-HT1B, 5-HT1D and 5-HT5A receptor expression in the stellate ganglion from normoglycaemic and diabetic rats. In the diabetic group, i) i.v. continuous infusions of 5-HT induced a cardiac sympatho-inhibition that was mimicked by the 5-HT1/5A agonist 5-carboxamidotryptamine (without modifying noradrenaline-induced tachycardia), but not by the agonists indorenate (5-HT1A), CP 93,129 (5-HT1B), PNU 142633 (5-HT1D), or LY344864 (5-HT1F); ii) SB 699551 (5-HT5A antagonist; i.v.) completely reversed 5-CT-induced cardiac sympatho-inhibition; and iii) 5-HT5A receptors were more expressed in the stellate ganglion compared to normoglycaemic rats. These results show the prominent role of the peripheral 5-HT5A receptors prejunctionally inhibiting the cardiac sympathetic drive in type 1 diabetic rats.

Serotonin-2B receptor antagonism increases the activity of dopamine and glutamate neurons in the presence of selective serotonin reuptake inhibition.

Previous research has implicated the serotonin-2B (5-HT2B) receptor as a possible contributor to the antidepressant-like response. Aripiprazole has been successfully used in combination with selective serotonin reuptake inhibitors (SSRIs) in treatment-resistant depression and it, among all receptors, exhibits the highest affinity for the 5-HT2B receptor. However, the potential contribution of such an antagonistic action on 5-HT2B receptors in the context of adjunct therapy is not known. In vivo electrophysiological recordings of ventral tegmental area (VTA) dopamine (DA) neurons, dorsal raphe nucleus (DRN) 5-HT neurons and pyramidal neurons in the medial prefrontal cortex (mPFC), and the hippocampus were conducted in anaesthetized Sprague-Dawley rats after the administration of 5-HT2B receptor ligands alone or in combination with the SSRI escitalopram. An escitalopram-induced decrease in DA, but not 5-HT firing activity, was rescued by 2-day co-administration of the selective 5-HT2B receptor antagonist LY266097. In the mPFC, 14-day escitalopram administration alone had no effect on pyramidal neuron firing and burst activity, whereas, aripiprazole administered alone or in combination with escitalopram for 14 days increased pyramidal neuron firing and burst activity. Likewise, the administration of LY266097 alone or its addition on the last 3 days of a 14-day escitalopram regimen increased pyramidal neuron firing and burst activity. These results indicated that 5-HT2B receptors play, at least in part, a role in this enhancement. In the hippocampus, 5-HT2B receptor activation by BW723c86 decreased escitalopram-induced inhibition of 5-HT reuptake, which was reversed by a 5-HT2B receptor antagonist. Altogether, these results put into evidence the possibility that 5-HT2B receptor blockade contributes to the therapeutic effect of aripiprazole addition to SSRIs in depression.

Receptor Interaction Profiles of 4-Alkoxy-Substituted 2,5-Dimethoxyphenethylamines and Related Amphetamines.

Background: 2,4,5-Trimethoxyamphetamine (TMA-2) is a potent psychedelic compound. Structurally related 4-alkyloxy-substituted 2,5-dimethoxyamphetamines and phenethylamine congeners (2C-O derivatives) have been described but their pharmacology is mostly undefined. Therefore, we examined receptor binding and activation profiles of these derivatives at monoamine receptors and transporters. Methods: Receptor binding affinities were determined at the serotonergic 5-HT1A, 5-HT2A, and 5-HT2C receptors, trace amine-associated receptor 1 (TAAR1), adrenergic α1 and α2 receptors, dopaminergic D2 receptor, and at monoamine transporters, using target-transfected cells. Additionally, activation of 5-HT2A and 5-HT2B receptors and TAAR1 was determined. Furthermore, we assessed monoamine transporter inhibition. Results: Both the phenethylamine and amphetamine derivatives (K i = 8–1700 nM and 61–4400 nM, respectively) bound with moderate to high affinities to the 5-HT2A receptor with preference over the 5-HT1A and 5-HT2C receptors (5-HT2A/5-HT1A = 1.4–333 and 5-HT2A/5-HT2C = 2.1–14, respectively). Extending the 4-alkoxy-group generally increased binding affinities at 5-HT2A and 5-HT2C receptors but showed mixed effects in terms of activation potency and efficacy at these receptors. Introduction of a terminal fluorine atom into the 4-ethoxy substituent by trend decreased, and with progressive fluorination increased affinities at the 5-HT2A and 5-HT2C receptors. Little or no effect was observed at the 5-HT1A receptor for any of the substances tested (K i ≥ 2700 nM). Phenethylamines bound more strongly to the TAAR1 (K i = 21–3300 nM) compared with their amphetamine analogs (K i = 630–3100 nM). Conclusion: As seen with earlier series investigated, the 4-alkyloxy-substituted 2,5-dimethoxyamphetamines and phenethylamines share some trends with the many other phenethylamine pharmacophore containing compounds, such as when increasing the size of the 4-substituent and increasing the lipophilicity, the affinities at the 5-HT2A/C subtype also increase, and only weak 5-HT2A/C subtype selectivities were achieved. At least from the binding data available (i.e., high affinity binding at the 5-HT2A receptor) one may predict mainly psychedelic-like effects in humans, at least for some of the compound investigated herein.

Cervical spinal 5-HT2A and 5-HT2B receptors are both necessary for moderate acute intermittent hypoxia-induced phrenic long-term facilitation.

Serotonin (5-HT) is a key regulator of spinal respiratory motor plasticity. For example, spinal 5-HT receptor activation is necessary for the induction of phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity triggered by moderate acute intermittent hypoxia (mAIH). mAIH-induced pLTF is blocked by cervical spinal application of the broad-spectrum 5-HT-receptor antagonist, methysergide. However, methysergide does not allow distinctions between the relative contributions of different 5-HT receptor subtypes. Intravenous administration of the Gq protein-coupled 5-HT2A/2C receptor antagonist ketanserin blocks mAIH-induced pLTF when administered before, but not after, mAIH; thus, 5-HT2 receptor activation is necessary for the induction but not maintenance of mAIH-induced pLTF. However, systemic ketanserin administration does not identify the site of the relevant 5-HT2A/2C receptors. Furthermore, this approach does not differentiate between the roles of 5-HT2A versus 5-HT2C receptors, nor does it preclude involvement of other Gq protein-coupled metabotropic 5-HT receptors capable of eliciting long-lasting phrenic motor facilitation, such as 5-HT2B receptors. Here we tested the hypothesis that mAIH-induced pLTF requires cervical spinal 5-HT2 receptor activation and determined which 5-HT2 receptor subtypes are involved. Anesthetized, paralyzed, and ventilated adult male Sprague Dawley rats were pretreated intrathecally with cervical (~C3-C5) spinal injections of subtype selective 5-HT2A/2C, 5-HT2B, or 5-HT2C receptor antagonists before mAIH. Whereas cervical spinal 5-HT2C receptor inhibition had no impact on mAIH-induced pLTF, pLTF was no longer observed after pretreatment with either 5-HT2A/2C or 5-HT2B receptor antagonists. Furthermore, spinal pretreatment with an MEK/ERK MAPK inhibitor blocked phrenic motor facilitation elicited by intrathecal injections of 5-HT2A but not 5-HT2B receptor agonists. Thus, mAIH-induced pLTF requires concurrent cervical spinal activation of both 5-HT2A and 5-HT2B receptors. However, these distinct receptor subtypes contribute to phrenic motor facilitation via distinct downstream signaling cascades that differ in their requirement for ERK MAPK signaling. The demonstration that both 5-HT2A and 5-HT2B receptors make unique contributions to mAIH-induced pLTF advances our understanding of mechanisms that underlie 5-HT-induced phrenic motor plasticity.NEW & NOTEWORTHY Moderate acute intermittent hypoxia (mAIH) triggers a persistent enhancement in phrenic motor output, an effect termed phrenic long-term facilitation (pLTF). mAIH-induced pLTF is blocked by cervical spinal application of the broad-spectrum serotonin (5-HT) receptor antagonist methysergide, demonstrating the need for spinal 5-HT receptor activation. However, the exact type of 5-HT receptors required for initiation of pLTF remains unknown. To the best of out knowledge, the present study is the first to demonstrate that 1) spinal coactivation of two distinct Gq protein-coupled 5-HT2 receptor subtypes is necessary for mAIH-induced pLTF, and 2) these receptors contribute to pLTF via cascades that differ in their requirement for ERK MAPK signaling.

5-HT1B Receptor-Mediated Activation of ERK1/2 Requires Both Gαi/o and β-Arrestin Proteins.

5-HT1B receptors modulate synaptic serotonin (5-HT) levels and play a significant role in the regulation of emotional behaviors. These receptors are Gαi/o-coupled and inhibit adenylyl cyclase but have also been reported to activate MAP kinases; however, the details of signaling cascades downstream of 5-HT1B receptor activation remain unclear, particularly in neuronal cells. We generated a stable 5-HT1B receptor-expressing Neuro2A (N2A-1B) neuronal cell line and demonstrate that activation of these receptors by the selective 5-HT1B agonist CP-94253 results in activation of ERK1/2 but not of other closely related MAP kinases. Phosphoproteomics revealed four novel phosphorylation sites on the third intracellular loop of the 5-HT1B receptor, and mutations of serine-256 and serine-291 to alanine led to reduced levels of ERK1/2 phosphorylation following receptor activation. Inhibition of Gαi/o signaling with pertussis toxin, as well as MEK1/2 inhibition with U0126, also reduced 5-HT1B-mediated ERK1/2 phosphorylation. Finally, we found that knockout of either β-arrestin 1 or β-arrestin 2 prevented 5-HT1B-mediated phosphorylation of ERK1/2. Taken together, these results show that 5-HT1B receptor activation selectively induces ERK1/2 activation through both the Gαi subunit and β-arrestin proteins. This work elucidates the signal transduction pathway of 5-HT1B receptors, as well as key phosphorylation sites within the receptor that modulate ERK1/2 activation, and further characterizes the intracellular mechanisms that underlie 5-HT1B receptor function.

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 μM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.