Because of its exquisite sensitivity to serotonin, the rat stomach fundus was used as a bioassay for serotonin before the development of more quantitative analytical assays for this biogenic amine (Vane 1957). Although the potency for the contractile effects of serotonin has been known for long time, the receptors mediating such a response have eluded definitive characterization for long time. Pharmacological studies attempting to characterize the contractile serotonergic receptor in the rat stomach fundus documented the similarity to the 5-HT2C receptor. In the absence of detectable 5-HT2C receptor mRNA in the rat stomach fundus, only molecular cloning allowed the identification of a new receptor in 1992 in rat and mouse (Foguet et al. 1992; Kursar et al. 1992; Loric et al. 1992; Wainscott et al. 1993) and in 1994 in humans (Choi et al. 1994; Kursar et al. 1994; Schmuck et al. 1994; Wainscott et al. 1996), now called 5-HT2B receptor. Subsequent pharmacological characterization of this receptor subtype in various species identified differences in its pharmacology and confirmed the close identity of this pharmacology to that of 5-HT2C receptors. Its physiological and pathophysiological functions include many differentiation steps both in periphery and central nervous system that were not previously identified or attributed to other receptor subtypes.
Structure and Properties of 5-HT2B Receptors
BW723C86: 1-methyl-2- [5-(2-thienylmethoxy)-1H-indole-3-yl] ethylamine hydrochloride has been reported to have 10-fold selectivity over the human 5-HT2C and 100-fold selectivity over the 5-HT2A receptors. Recommended use: ≤100 nM concentration or ≤3 mg/kg ip in rodents (Porter et al. 1999; Jerman et al. 2001; Knight et al. 2004; Cussac et al. 2008).
Alpha-methyl serotonin is a full agonist with high affinity for the 5-HT2B receptor site (pEC50=8.4) and lower affinity for both 5-HT2C and 5-HT2A.
5-Methoxytryptamine is also 25- and 400-fold selective over the 5-HT2A and 5-HT2C receptor sites, respectively.
Nor-dexfenfluramine (metabolite of dexfenfluramine), methylergonovine (metabolite of methysergide), and Ro 60-0175: 2(S)-1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate are all preferential 5-HT2B agonists with about 10-fold selectivity over 5-HT2C receptor.
Pharmacological analysis has shown that the 5-HT2B receptor displays high affinity binding to its endogenous ligand serotonin (Kd ~10 nM), a value significantly different from that for the 5-HT2A receptor. 5-HT2B receptors are the targets of many 5-HT2 nonselective compounds, metabolites of therapeutic compounds, and drugs of abuse. Agonists include MDA (3,4-methylene dioxyamphetamine-MDA, metabolite of 3,4-Methylenedioxy methamphetamine-MDMA) (Setola et al. 2003), tryptamine, and lysergic acid diethylamide (LSD) all exhibit the highest potencies for 5-HT2B. 2,5-dimethoxy-4-iodoamphetamine (DOI) is a nearly full agonist at 5-HT2B receptors with similar affinity to 5-HT2A and 5-HT2B receptors (Porter et al. 1999; Jerman et al. 2001; Knight et al. 2004; Cussac et al. 2008). Many substances from the class of “new” drugs known as “legal highs” were found to display notable affinity for 5-HT2B receptors, including 5-APB (Ki = 14 nM) and 6-APB (Ki = 3.7 nM), and 5-iodo-aminoindane (Ki = 70 nM). Functional assays of 5-APB and 6-APB confirmed that these compounds acted as potent (i.e., nanomolar EC50 values) full agonists at 5-HT2B receptors (Iversen et al. 2013; Rickli et al. 2015). Other compounds exhibited submicromolar affinities for the 5-HT2B receptor (mephedrone, naphyrone, 1-naphyrone, and methylenedioxy-aminotetralin). 5-APB, commonly marketed as “benzofury” a new psychoactive substance, was shown to cause contraction of rat stomach fundus, which was reversed by the 5-HT2B receptor antagonist RS127445 (Dawson et al. 2014). This finding is potentially important, because previous studies have shown that there was a correlation in a series of phenyliso-propylamines between hallucinogenic activity and affinity for the 5-HT2B receptor (Nelson et al. 1999). Activation of this receptor appears to play a key role in the behavioral stimulant and serotonin releasing effects of MDMA (Doly et al. 2008) and in the reinforcing effects of MDMA in mice (Doly et al. 2009).
The first highly selective antagonist reported was LY266097: 1-(2-chloro-3,4- dimethoxybenzyl)- 6-methyl- 1,2,3,4-tetrahydro- 9Hpyrido [3,4-b]indole hydrochloride with a pKi of 9.7 for the human cloned 5-HT2B receptor and a 100-fold greater selectivity over human 5-HT2C and 5-HT2A sites (Audia et al. 1996). Recommended use: 20 nM concentration or 0.5 mg/kg ip in rodents.
SB204741: N-(1-methyl-5-indolyl)-N′-(3-methyl-5-isothiazolyl)urea has been reported as a selective 5-HT2B receptor antagonist with approximately 100-fold selectivity over the 5-HT2C and 5-HT2A sites but with quite low potency (Ki around 100 nM) (Bonhaus et al. 1995). Recommended use: 500 nM concentration or 10 mg/kg ip in rodents.
The tetrahydro-β-carboline, LY272015: 6-chloro-5-methyl -N-(5-quinolinyl) -2,3-dihydro -1H-indole-1-carboxamide is also a fairly selective and highly potent antagonist (Cohen et al. 1996). Recommended use: 50 nM concentration or 1 mg/kg ip in rodents.
RS127445: 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine was found to have subnanomolar affinity for the 5-HT2B receptor (pKi = 9.5) and 1000-fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites and appears as the most selective, high affinity 5-HT2B receptor antagonist suitable now (Bonhaus et al. 1999). Recommended use: 20 nM concentration or 0.25 mg/kg ip in rodents.
Recently, the methoxythioxanthene BF-1 was reported as a highly selective and potent 5-HT2B receptor antagonist lacking high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2, as well as muscarinic M1 and M2 receptors (Schmitz et al. 2015).
S33526: 6-chloro-2,3,4,9-tetrahydro-1H-b-carbolin-1-yl)-phenyl-acetic acid ethyl ester, behaves as high affinity and relatively selective antagonist at 5-HT2B receptors (Cussac et al. 2002).
SB215505: 6-chloro-5-methyl-N-(5-quinolinyl)-2,3-dihydro-1H-indole-1-carboxamide behaves as high affinity and preferential inverse agonists at 5-HT2B receptors.
SB206553: 5-methyl-N-(3-pyridyl)-1,2,3,5-tetrahydrobenzo[1,2-b:4,5-b’]dipyrrole-1-carboxamide is a mixed 5-HT2C/5-HT2B receptor antagonist. It has been reported as a selective 5-HT2C/2B receptor inverse agonist with 50- to 100-fold lower affinity for the 5-HT2A and other sites.
Nonselective 5-HT2 receptor antagonists such as ritanserin and metergoline antagonize 5-HT2B receptor-mediated effects. Also, the α2 adrenergic receptor antagonists yohimbine and rauwolscine are potent 5-HT2B antagonists and have a low affinity for the 5-HT2C and 5-HT2A receptor sites. Atypical antipsychotics have also fairly high affinity for 5-HT2B receptors including clozapine, asenapine, or cariprazine (Wainscott et al. 1996; Millan et al. 2003; Shahid et al. 2009; Kiss et al. 2010). Aripiprazole (OPC-14597) is a new generation atypical antipsychotic drug, which has high antagonist affinity (EC50 = 11 nM) for the human 5-HT2B receptor (Shapiro et al. 2003).
The 5-HT2B receptor is a 7 TM receptor, with fairly long N-terminus of about 55 amino acids. A consensus site of N-glycosylation can be found that is missing in the rat 5-HT2B receptor, questioning the N-glycosylation of the N-terminus of this receptor. The receptor consists of 481 AA in human and 479 AA in rat or mouse, with 79% homology for human versus rat and 82% homology for human versus mouse. A new and unanticipated role of the 5-HT2B receptor N-terminus as a negative modulator, affecting both constitutive and agonist-stimulated activity of the receptor, has been recently evidenced (Belmer et al. 2014).
The recently published 5-HT2B receptor bound to ergotamine crystal structure showed that it exhibits conformational characteristics of both the active and inactive states: an active-like state in the helix VII conformation of the 5-HT2B receptor, but only partial changes in helix VI, mirrored the strong β-arrestin bias of ergotamine at 5-HT2B receptors observed in pharmacological assays. The differential signaling patterns were also mirrored in the crystal structures, which showed features of a β-arrestin-biased activation state for the 5-HT2B receptor (Wacker et al. 2013; Wang et al. 2013). A likely structural explanation for the distinct conformational features and biased pharmacology of ergotamine for 5-HT2B receptors can be found in the region of the extracellular loop 2 (ECL2) junction with helix V, E212-R213-F214 forming an additional helical turn stabilized by a structured water molecule at the extracellular tip of helix V. The segment of ECL2 connecting helices III and V via the conserved disulfide bond is, therefore, shortened in the 5-HT2B receptor and creates a conformational constraint on the position of the extracellular tip of helix V (Martí-Solano et al. 2014). However, this structured water molecule involved in ECL2 junction with helix V has been challenged since differential interactions of ergotamine with the top of helices V and VI could determine the rotational freedom of helix VI (Liu et al. 2013).
No allosteric modulator has been definitely identified yet. Ergotamine has been shown to occupy two distinct sites in 5-HT2B receptors, the orthosteric site, where the indole nucleus of ergotamine resides, and the extended binding site, where the tripeptide portion is engaged. The allosteric site in the muscarinic M2 receptor is the same extracellular region as the tripeptide portion of ergotamine. These similarities in both the M2 and 5-HT2B receptors suggest that the location of the extracellular allosteric site for Class A GPCRs is quite similar and, in fact, argue that ergotamine likely functions as a bitopic ligand; that is, it occupies both the orthosteric and putative extracellular allosteric site in the 5-HT2B receptor. It is now thought that a sodium ion allosterically alters the binding pocket to dampen G-protein signaling, leaving β-arrestin recruitment intact. Structural consideration support that this sodium pocket is collapsed in the 5-HT2B receptor structure (McCorvy and Roth 2015).
Heteromeric Receptor Associations
In cardiac fibroblasts, AT1 angiotensin receptors and 5-HT2B receptors, which have been reported to share common signaling pathways, support a possible direct interaction between 5-HT2B and AT1 receptors. Using co-immunolocalization and a pull-down assay, the two receptors were shown to interact together, which suggested that these receptors could exist in heterodimeric complexes (Jaffre et al. 2009), but experimental confirmation is still lacking.
In Transfected Cells
The 5-HT2B receptor, when stably transfected in mouse fibroblast L-cells, has been shown to activate GTPase activity and inositol 1,4,5- triphosphate production upon agonist stimulation, which could be blocked by antibodies against Gαq/11, but not by pertussis or cholera toxins or by anti-Gαi or anti-Gαs antibodies. This GTPase activation was thus mediated by the G-protein Gαq/11, but not by Gαs or Gαi. The GTPase activation was also blocked by anti-β1–4 or -γ2 subunit antibodies. Agonist stimulation of the 5-HT2B receptor caused a rapid and transient activation of the proto-oncogene product p21ras in response to serotonin, as measured by an increase in GTP bound-Ras (Launay et al. 1996). Furthermore, 5-HT2B receptor stimulation activated the mitogen-activated protein kinases (MAPKs) p42mapk/p44mapk , extracellular signal-regulated kinase 2 and 2 (ERK2/ERK1). In addition to a mitogenic action, a transforming activity of serotonin was mediated by the 5-HT2B receptor as it led to the formation of foci and to the formation of tumors from these foci in nude mice (Launay et al. 1996). Moreover, the 5-HT2B receptor-dependent cell cycle progression occurred through retinoblastoma protein hyperphosphorylation and the activation of both cyclin D1/cdk4 and cyclin E/cdk2 kinases. The induction of cyclin D1 expression, but not that of cyclin E, was under MAPK control, indicating an independent regulation of these two cyclins in 5-HT2B receptor mitogenesis. Platelet-derived growth factor receptor (PDGFR) kinase activity was essential for 5-HT2B-triggered MAPK/cyclin D1, but not cyclin E, signaling pathways (Nebigil et al. 2000b). The 5-HT2B receptor activation also increases activity of the Src family kinases c-Src, Fyn, and c-Yes. Strikingly, c-Src, but not Fyn or c-Yes, was the crucial molecule between the Gq protein-coupled 5-HT2B receptor and the cell cycle regulators and inhibition of c-Src activity was sufficient to abolish the serotonin-induced: (i) PDGFR tyrosine kinase phosphorylation and MAPK activation, (ii) cyclin D1 and cyclin E expression levels, and (iii) thymidine incorporation. Thus, c-Src activation by the 5-HT2B receptor controlled cyclin E induction and, in concert with the receptor tyrosine kinase PDGFR, induced cyclin D1 expression via the MAPK/ERK pathway (Nebigil et al. 2000b). The 5-HT2B receptor is also coupled to the phospholipase A2 (PLA2)-mediated release of arachidonic acid (Tournois et al. 1998). In addition, stimulation of the 5-HT2B receptor triggered intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the carboxy terminus of the 5-HT2B receptor was shown to be required for recruitment of the cNOS transduction pathways, and iNOS stimulation was under control of the Gα13 pathways (Manivet et al. 2000). The 5-HT2B receptor shares the C-terminal E-X-V/I-S-X-V sequence with 5-HT2C receptors and also binds MUPP1-PDZ domains in-vitro (Becamel et al. 2001).
In Primary Cultures
In primary culture of cardiac fibroblasts, angiotensin II (AngII) or serotonin-dependent cytokine release processes were critically dependent on HB-EGF and Src activition via endogenous AT1 and 5-HT2B receptors (Jaffre et al. 2009). Matrix metalloproteinases (MMPs) were responsible for HB-EGF shedding and subsequent EGF-receptor transactivation that was induced by AngII or serotonin. Tumor necrosis factor-alpha (TNF-α) converting enzyme (TACE, ADAM-17) was found to control HB-EGF shedding in fibroblasts and was directly regulated by 5-HT2B receptors (Pietri et al. 2005). All these findings support the idea that AT1 and 5-HT2B receptors share common EGF receptor-dependent signaling pathways in adult cardiac fibroblasts. Blockade of one of the two receptors prevented cytokine release induced by the other receptor, supporting the idea of interactions between 5-HT2B and AT1 receptors (Jaffre et al. 2009). Using co-immunolocalization and a pull-down assay, the two receptors were shown to interact in a common cell compartment. Reports have suggested that these receptors exist in heterodimeric complexes that may play a key role in receptor maturation and trafficking to the plasma membrane and/or signaling (Bulenger et al. 2005). Together, these findings indicate that heteromeric AT1 and 5-HT2B receptors exist and result in common signaling complexes regulating hypertrophic factors in heart (Jaffre et al. 2009).
Stimulation of 5-HT2B receptors on hepatic stellate cells (HSC) was recently shown to activate expression of TGFβ1 (a powerful suppressor of hepatocyte proliferation) via ERK/JunD signaling. Antagonists of 5-HT2B receptor have been shown to decrease mRNA levels of TGFβ1, connective growth factor, plasminogen activator inhibitor-1, Smad-3, and JunD in lung and skin fibroblasts (Dees et al. 2011). Activation of the 5-HT2B receptor leads to sustained phosphorylation of two downstream targets of mTOR, p70S6K, and 4E–BP1, thereby facilitating survival and inhibiting autophagy of hepatocellular carcinomas (Soll et al. 2010).
The 5-HT2B receptor has been shown to protect newborn postmitotic cardiomyocytes against serum deprivation-induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation, and TUNEL labeling. Serotonin prevented cytochrome c release and caspase-9 and -3 activation after serum deprivation via cross-talks between phosphatidylinositol-3 kinase (PI3K)/Akt and ERK1/2 signaling pathways. Serotonin binding to 5-HT2B receptor activated ERK kinases that inhibited Bax expression induced by serum deprivation. Serotonin via PI3K/Akt activated nuclear factor-κB (NF-κB) that was required for the regulation of the mitochondrial adenine nucleotide translocator (ANT-1) and mitochondrial permeability. These findings identified serotonin as a novel cardiomyocyte survival factor targeting mitochondria (Nebigil et al. 2003). Interestingly, NF-κB regulation by 5-HT2B receptors was confirmed in a large screen for genes regulating NF-κB and the MAPK pathways (Matsuda et al. 2003).
The 5-HT2B receptor mRNA was detected in mouse embryos since 8.5 days postcoitum (dpc), whereas there were only low levels of 5-HT2A and no 5-HT2C receptor mRNA expression at this stage. Expression of this receptor was confirmed by pharmacological assays (Choi et al. 1997). Teratogenesis by retinoic acid or blockade of serotonergic signaling by 5-HT2 receptor antagonists perturbs development, resulting in forebrain and olfactory placode anomalies, malformations of the face, eye, and lens, as well as posterior neural tube and cardiac defects (Bhasin et al. 2004). These pathways may act as opposing signals for common targets in the mouse embryo. The importance of 5-HT2B receptors in cardiac development has been validated by genetic inactivation by homologous recombination of the 5-HT2B receptor gene, which leads to embryonic and neonatal death due to the following defects in the heart: Htr 2B −/− embryos exhibit a lack of trabeculae in the heart leading to mid-gestation lethality, and newborn Htr 2B −/− mice exhibit cardiac dilation resulting from contractility deficits and structural deficits at the intercellular junctions between cardiomyocytes (Nebigil et al. 2000a). Report showed that serotonin acts downstream of lactogen signaling to stimulate β-cell proliferation. Blocking 5-HT2B signaling in pregnant mice also blocked β-cell expansion and caused glucose intolerance (Kim et al. 2010).
By Northern blot and immunohistochemistry, 5-HT2B receptor mRNA and protein expression was detected in the stomach fundus, intestine, liver, kidney, pancreas, spleen, and lung, as well as in the myocardium of several species including rats, mice, and humans (Kursar et al. 1992; Choi et al. 1994; Kursar et al. 1994; Choi and Maroteaux 1996). The presence of a functionally active 5-HT2B receptor subtype in human uterine smooth muscle cells was reported (Kelly and Sharif 2006). Reports using pharmacological or molecular studies indicated that the 5-HT2B receptor was also expressed in blood vessels, including smooth muscle cells (Ullmer et al. 1995) as well as on the endothelial cells of pig pulmonary arteries (Glusa and Pertz 2000), of human meningeal blood vessels (Schmuck et al. 1996), and of rat jugular vein (Ellis et al. 1995). The gene coding for the 5-HT2B receptor was found expressed in human and rodent anagen skin (but not telogen), in melanomas and Mel A melanocytes (Slominski et al. 2003; Slominski et al. 2004). In addition, 5-HT2B receptor expression has been detected in rat spleen, thymus, and peripheral blood lymphocytes (Stefulj et al. 2000). A 5-HT2B receptor expression was detected in c-kit+ bone-marrow cells (Launay et al. 2012). Finally, 5-HT2B receptor expression was also demonstrated in rat osteocytes, osteoblasts and a population of periosteal fibroblasts containing osteoblast precursor cells (Bliziotes et al. 2001; Westbroek et al. 2001).
In human brain, 5-HT2B receptor expression has been reported in the cerebral cortex, cerebellar nuclei and their projection areas, lateral septum, dorsal hypothalamus, and medial amygdala (Kursar et al. 1992; Choi et al. 1994; Kursar et al. 1994; Choi and Maroteaux 1996). Expression of serotonin 5-HT2B receptor mRNA was also confirmed in several brain nuclei including the dorsal raphe nuclei by gene expression profiling in the mammalian brain and by in-situ hybridization (Bonaventure et al. 2002). Human brain expression was confirmed by qPCR in frontal, temporal, parietal, and occipital lobe, olfactory region, cerebellum, diencephalon, hippocampus, thalamus, pituitary gland, pons, medulla oblongata, and nucleus accumbens (NAcc) (Bevilacqua et al. 2010). The presence of microglial 5-HT2B receptors was confirmed by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. Two photon microscopy on brain slices (Kolodziejczak et al. 2015) and patch-clamp experiments in cultured microglia (Krabbe et al. 2012) confirmed that modulation of microglial functions like phagocytosis and migration is fundamental for the central nervous system since microglia can influence the balance of synaptogenesis and neuronal death in pathology.
The 5-HT2B receptor has also been shown to participate in the release of rat growth hormone (GH) through a pituitary site since the 5-HT2B receptor agonist BW723C86 stimulated GH release that was blocked by the 5-HT2B receptor antagonist SB204741 (Papageorgiou and Denef 2007). The expression of 5-HT2B receptor mRNA was reported in rat cultured astrocytes (Sanden et al. 2000; Osredkar and Krzan 2009). Expression of the 5-HT2B receptor was also found in spinal cord tissue (Helton et al. 1994; Holohean and Hackman 2004), in the rat organ of Corti lateral wall and spiral ganglion subfractions (Oh et al. 1999), and upregulated with age in the mice cochlea (Tadros et al. 2007). In human eye, HTR 2B mRNA was found predominantly expressed in the retina, ciliary body, ciliary epithelium, choroid, conjunctiva, and iris. Optic nerve tissue of human donors exhibited the presence of HTR 2B mRNA as human trabecular meshwork cells (Sharif and Senchyna 2006).
Pathophysiological Functions in Central Nervous System
Response to Antidepressants
Neurotransmission by serotonin is tightly regulated by autoreceptors that fine-tune serotonergic neurotransmission through negative feedback inhibition at the cell bodies (predominantly 5-HT1A) or at the axon terminals (predominantly 5-HT1B); however, different roles for 5-HT2B receptors have also been detected (McDevitt and Neumaier 2011). The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking the SERT and rely on long-term adaptations of pre- and postsynaptic receptors. Long-term behavioral and neurogenic SSRI effects were abolished after either genetic or pharmacologic inactivation of 5-HT2B receptors (Diaz et al. 2012). Conversely, direct agonist stimulation of 5-HT2B receptors induced an SSRI-like response in behavioral and neurogenic assays. The 5-HT2B receptor was found expressed by raphe serotonergic neurons by single cell PCR. The SSRI-induced increase in hippocampal extracellular serotonin concentration was strongly reduced in the absence of functional 5-HT2B receptors. Furthermore, a selective 5-HT2B agonist mimicked SSRI responses, supporting a positive regulation of serotonergic neurons by 5-HT2B receptors (Diaz et al. 2012). Furthermore, Htr 2B −/− mice display an antidepressant-like phenotype, which includes reduced latency to feed in the novelty suppressed feeding test, basal increase in hippocampal BDNF levels, no change in TrkB and p75 protein levels, and an increased preference for sucrose consumption compared to wild-type mice. Nevertheless, these mice can develop depressive-like behaviors when socially isolated during 4 weeks. SSRI have been previously shown to be ineffective in nonstressed Htr 2B −/− mice and as well in chronically stressed Htr 2B −/− mice. No behavioral or plastic effect was induced by SSRI antidepressants (Diaz et al. 2016). The 5-HT2B receptor appears, therefore, to positively modulate serotonergic activity and to be required for the therapeutic actions of SSRIs.
Response to Anorexigens
The hypophagic response to the anorexigen and serotonin releaser, dexfenfluramine, observed in wild-type mice is also eliminated in Htr 2B −/− mice or in wild-type mice treated with RS127445. Using microdialysis, the dexfenfluramine-induced hypothalamic peak of serotonin release was found strongly reduced in Htr 2B −/− awake mice compared with wild type. Moreover, a strong serotonin release was observed upon dexfenfluramine stimulation of a synaptosomal preparation from wild type but not from Htr 2B −/− mice (Banas et al. 2011). A 5-HT2B receptor-dependent modulation of presynaptic effectors may explain this requirement for 5-HT2B receptors in releaser action.
Response to MDMA
The “club drug” 3,4-methylenedioxymethamphetamine (MDMA; also known as Ecstasy) binds preferentially to and reverses the activity of the serotonin transporter, causing release of serotonin stores from nerve terminals. Subsequent activation of postsynaptic 5-HT receptors by released 5-HT has been shown to be critical for the unique psychostimulatory effects of MDMA. Acute pharmacological inhibition or genetic ablation of the 5-HT2B receptor in mice completely abolishes MDMA-induced hyperlocomotion and 5-HT release in NAcc and ventral tegmental area. Furthermore, the 5-HT2B receptor dependence of MDMA-stimulated release of endogenous 5-HT from superfused midbrain synaptosomes suggests that 5-HT2B receptors act, unlike any other 5-HT receptor, presynaptically to affect MDMA-stimulated 5-HT release. Thus, the 5-HT2B receptor is a novel regulatory component in the actions of MDMA (Doly et al. 2008). However, the role of serotonin/dopamine interactions in the behavioral effects of MDMA remains unclear. Htr 2B −/− mice do not exhibit behavioral sensitization or conditioned place preference following MDMA injections. In addition, MDMA-induced reinstatement of conditioned place preference after extinction and locomotor sensitization development are each abolished by a 5-HT2B receptor antagonist (RS127445) in wild-type mice. Accordingly, MDMA-induced dopamine D1 receptor-dependent phosphorylation of extracellular-regulated kinase in NAcc is abolished in mice lacking functional 5-HT2B receptors. These results underpin the importance of 5-HT2B receptors in the reinforcing properties of MDMA and illustrate the importance of serotonin/dopamine interactions on MDMA effects (Doly et al. 2009). Moreover, another selective 5-HT2B receptor antagonist LY 266097, which has no influence on basal accumbal and striatal DA outflow, reduces significantly accumbal DA outflow. A significant reduction of basal DA outflow in the NAcc was also observed after i.p. administration of the 5-HT2B receptor antagonist RS 127445. In contrast, the 5-HT2B receptor agonist BW 723C86 had no influence on basal DA outflow in either brain region. The increase in striatal and accumbal DA outflow induced by the 5-HT2B/2C receptor inverse agonist SB 206553 was unaltered by LY 266097 pretreatment. Conversely, LY 266097 significantly diminished the increase in DA outflow induced by haloperidol or amphetamine in the NAcc, but not in the striatum. Amphetamine-induced hyperlocomotion was also attenuated by LY 266097. Thus 5-HT2B receptors exert a facilitatory control on mesoaccumbens DA pathway activity and may constitute a new target for improved treatment of DA-related neuropsychiatric disorders (Auclair et al. 2010).
These findings strongly suggest that activation of 5-HT2B receptors is a limiting step in the SERT-dependent increase in extracellular 5-HT that is required for SSRIs, MDMA, or anorexigen action.
Impulsivity and Psychosis
Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. A population-specific serotonin 2B (5-HT2B) receptor stop codon (i.e., HTR 2B Q20*) was reported to segregate with severely impulsive individuals (Bevilacqua et al. 2010). Especially under conditions where control was impaired, the carriers of the stop codon being more vulnerable to alcohol were more impulsive if they drank. Similarly, Htr 2B −/− mice displayed more impulsive choice in delayed discounting tasks, sought novelty, and were more active after receiving a D1 dopamine receptor agonist (Bevilacqua et al. 2010). Interestingly, in the same cohort, early onset schizophrenia was more prevalent in HTR 2B Q*20 carriers. Recently, it was shown that domains related to the positive, negative, and cognitive symptom-clusters of schizophrenia are affected in Htr 2B −/− mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr 2B −/− mice presented with enhanced locomotor response to the psychostimulant dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment (Pitychoutis et al. 2015). 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provides first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders.
The phenotype of Htr 2B −/− mice results from a combination of both the direct absence of 5-HT2B receptor signaling and the neural adaptations triggered by the permanent lack of this receptor.
Pathophysiological Peripheral Functions
Cardiopulmonary and Vascular Diseases
Inactivation of the 5-HT2B receptor gene by homologous recombination leads to partial embryonic lethality due to defects in the heart development. Neonates exhibit a second wave of partial lethality due to cardiac dilation resulting from contractility deficits and structural deficits at the intercellular junctions between cardiomyocytes. Echocardiography and electrocardiography studies in animals that past the first week and survive until adulthood confirm the presence of left ventricular dilation and decreased systolic function (Nebigil et al. 2000a). Serotonin, via the 5-HT2B receptor, regulates differentiation and proliferation during development as well as cardiac structure and function in adults. In adults, 5-HT2B receptors were found to be overexpressed in heart from patients with congestive heart failure (Jaffre et al. 2009). Serotonin plasma levels are also increased in patients with heart failure and in animal studies with cardiac hypertrophy induced by aortic constriction. By mimicking sympathetic stimulation in vivo, mice globally lacking serotonin 5-HT2B receptors did not develop isoproterenol-induced left ventricular hypertrophy (Jaffré et al. 2004). The exact cardiac cell type(s) expressing 5-HT2B receptors (cardiomyocytes versus noncardiomyocytes) involved in this pathological heart hypertrophy was addressed in vivo: mice expressing the 5-HT2B receptor solely in cardiomyocytes, like global 5-HT2B receptor-null mice, are resistant to isoproterenol-induced cardiac hypertrophy and dysfunction, as well as to isoproterenol-induced increases in plasma cytokine levels pointing to noncardiomyocytes-mediated effects (Jaffre et al. 2009). In primary culture of cardiac fibroblasts, angiotensin II and isoproterenol stimulated NOX activity that was prevented by a selective antagonist (SB215505). The 5-HT2B receptor blockade by SB215505 prevented the increase in cardiac superoxide generation and hypertrophy in two models of cardiac hypertrophy, i.e., angiotensin II and isoproterenol infusions in mice (Monassier et al. 2008). A functional interaction between AT1 and 5-HT2B receptors via a transinhibition mechanism that may involve heterodimeric receptor complexes was shown to trigger cytokine release in cardiac fibroblasts (Jaffre et al. 2009).
The 5-HT2B receptor has also been shown to be involved in cardiac hypertrophy by acting directly on cardiac myocytes. After 2 weeks of aortic banding surgery, mRNA and protein expression of 5-HT2B receptors increased significantly. The antagonist, SB215505, significantly reduced the increase in heart weight, heart wall thickness, left ventricular mass, and the expression of the brain natriuretic peptide (BNP) but did not attenuate the upregulation of 5-HT2B receptor protein expression in rats after aortic banding. Following in-vitro mechanical stretch of cardiomyocytes and incubation with serotonin, the level of 5-HT2B receptors and BNP protein increased time-dependently. When transfected with specific siRNA for 5-HT2B receptors in cardiomyocytes, the increase of NF-κB translocation and BNP protein induced by serotonin incubation plus mechanical stretch were both reversed (Liang et al. 2006). The involvement of 5-HT2B receptors was also reported in the generation of apoptotic events associated with cardiac remodeling during increased adrenergic stimulation (Bai et al. 2010).
These data revealed a dual role of 5-HT2B receptors on both cardiomyocytes and cardiac fibroblasts in regulating cardiac hypertrophy in vivo.
Pulmonary arterial hypertension (PAH) is a progressive and often fatal disorder in humans that results from an increase in pulmonary blood pressure associated with abnormal vascular proliferation. Serotonin is associated with the pathogenesis of PAH (Chan and Loscalzo 2008). Therapeutic drugs with PAH as a side effect, like the amphetamine derivative and anorexigen dexfenfluramine, are potent serotonin releasers acting at SERT and/or agonists at 5-HT2B receptors (Weir et al. 2008). Serotonergic anorexigen-induced PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Blockade of 5-HT2B receptors using independent approaches, either genetic (Htr 2B −/− ) or pharmacologic inactivation (5-HT2B receptor antagonist RS127445), completely prevented the development of hypoxia-induced pulmonary hypertension in mice including the increase in pulmonary blood pressure and lung remodeling, the increase in vascular proliferation, elastase activity, and transforming growth factor-β-1 (TGFβ1) levels (Launay et al. 2002). Using the monocrotaline-induced pulmonary hypertension model, more studies confirmed that other 5-HT2B receptor antagonists (terguride, PRX-08066, or C-122) significantly reduced pulmonary pressure, arterial wall thickening, and lumen occlusion but maintained cardiac function (Porvasnik et al. 2010; Dumitrascu et al. 2011; Zopf et al. 2011). Pulmonary hypertension is associated with a substantial increase in 5-HT2B receptor expression in pulmonary arteries in rodents and humans (Launay et al. 2002; Dumitrascu et al. 2011). Activation of 5-HT2B receptors appears to be, therefore, a limiting step in the development of pulmonary hypertension.
In Bmpr2R899X mutant mice, which spontaneously develop pulmonary hypertension, the 5-HT2B receptor antagonist, SB204741, prevented the development of pulmonary hypertension, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels (West et al. 2016). Using bone-marrow transplantation, the restricted expression of 5-HT2B receptors to bone-marrow cells was shown as necessary and sufficient for hypoxia- or monocrotaline-induced pulmonary hypertension to develop via an action at hematopoietic stem cell differentiation (Launay et al. 2012). Interestingly, Bmpr2R899X mutant bone-marrow cells were also shown to cause spontaneous pulmonary hypertension with remodeling and inflammation when transplanted into control mice, while control bone-marrow cells had a protective effect against the development of disease when transplanted into mutant mice. Bone-marrow cells play thus a key role in genetic pulmonary hypertension pathogenesis (Yan et al. 2016).
These findings reveal the limiting role of serotonin via 5-HT2B receptors in PAH development and shift the contribution of serotonin to PAH to an extrapulmonary, hematopoietic event.
One of the most profound increases in vascular responsiveness in hypertension has been observed for serotonin. Mesenteric arteries from deoxycorticosterone- (DOCA) salt hypertensive predominantly contracted via 5-HT2B receptors. PCR analyses indicated an increase in mRNA for the 5-HT2B receptor in mesenteric arteries of DOCA-salt hypertensive arteries, supporting an increase in receptor number (Watts et al. 1996). The endothelium-denuded isolated superior mesenteric artery of DOCA-salt rats displayed a marked increase in maximum contraction to the 5-HT2B receptor agonist BW723C86 compared with that of arteries from sham rats, confirming that the 5-HT2B receptor plays a greater role in serotonin-induced contraction in arteries from DOCA-salt rats. In chronically instrumented rats, the 5-HT2B-receptor antagonist LY272015 significantly reduced mean blood pressure (Watts and Fink 1999). LY272015 produced a fourfold rightward shift to serotonin in aorta from hypertensive rats made by exposure to the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine, and blockade by ketanserin did not occur at low concentrations of serotonin (Russell et al. 2002).
These findings revealed that the 5-HT2B receptor plays an important role in serotonin-induced contraction in arteries of hypertensive individuals.
Serotonin was shown to increase proliferation and collagen synthesis by lung fibroblasts. Serotonin concentrations in lung homogenates increased significantly over the time course of bleomycin-induced fibrosis, with a maximum at day seven, together with the expression of serotonin receptors 5-HT2A and 5-HT2B (Königshoff et al. 2010). Blockade of 5-HT2B receptors by SB215505 reduced bleomycin-induced lung fibrosis, as demonstrated by reduced lung collagen content and reduced procollagen 1 and procollagen 3 mRNA expression. 5-HT2B receptor antagonists promoted an antifibrotic environment by decreasing the lung mRNA levels of TGFβ1, connective growth factor and plasminogen activator inhibitor-1, and JunD mRNA. Interestingly, the 5-HT2B receptor was strongly overexpressed by fibroblasts in the fibroblastic foci of human idiopathic pulmonary fibrosis samples (Fabre et al. 2008). Serotonin contribution to lung fibrosis is thus controlled by 5-HT2B receptors regulating TGFβ1 levels.
In the liver, fibrogenic hepatic stellate cells (HSC) are known to express also 5-HT2A and 5-HT2B receptors that may regulate TGFβ1 and Smads (Li et al. 2006). After HSC activation, expression of 5-HT2A and 5-HT2B receptors was found 100- and 50-fold that of quiescent cells, respectively. The 5-HT2B receptor expression level was strongly associated with fibrotic tissue in diseased liver. Treatment of HSCs with 5-HT2 receptor antagonists suppressed proliferation and elevated their rate of apoptosis. Serotonin synergized with platelet-derived growth factor to stimulate increased HSC proliferation (Ruddell et al. 2006). Distinct from quiescent cells, activated HSCs exhibited [Ca2+]i transients following treatment with serotonin (Park et al. 2011). Stimulation of 5-HT2B receptors on HSC by serotonin was shown to activate expression of TGFβ1 via ERK/JunD signaling. Selective antagonism of 5-HT2B receptors enhanced hepatocyte growth in models of acute and chronic liver injury. Similar effects were observed in mice lacking 5-HT2B receptor or JunD and when HSCs have been selectively depleted (Ebrahimkhani et al. 2011). Therefore, 5-HT2B receptor appears to have a dual role on liver, promoting regeneration in physiological conditions and fibrosis in pathological conditions.
Dermal fibrosis was independently shown to be reduced in Htr 2B −/− mice using both inducible and genetic models of fibrosis. Pharmacologic inactivation of 5-HT2B receptor also effectively prevented the onset of experimental fibrosis and ameliorated established fibrosis by decreasing mRNA levels of TGFβ1, connective growth factor, plasminogen activator inhibitor-1, and Smad-3 (Dees et al. 2011). Serotonin contribution to skin fibrosis is thus controlled by 5-HT2B receptors via regulation of TGFβ1 levels.
Treatment of neonatal rat cardiac fibroblasts with serotonin increased the expression of smooth muscle α-actin, a marker of fibroblast differentiation into myofibroblasts and stimulated cardiac fibroblast migration. Incubation of cardiac fibroblasts with serotonin enhances secretion of TGFβ1 and expression of metalloproteinases (MMPs). Serotonin can thus directly act at neonatal cardiac fibroblasts by enhancing secretion of TGFβ1 and MMPs and promoting their migration and differentiation, which seem mediated through 5-HT2A receptors (Yabanoglu et al. 2009). Independently, serotonin- or AngII-stimulated cytokine release in adult cardiac fibroblasts was found to be sensitive to 5-HT2B receptor blockade, including secretion of TGFβ1. Treatments with epidermal growth factor receptor (EGFR, ErbB1/4)-selective inhibitors or with selective inhibitors of MMPs also abolished AngII- and serotonin-induced cytokine release. Finally, the use of HB-EGF −/− cardiac fibroblasts confirmed that EGFR stimulation is absolutely required for AngII- and serotonin-dependent cytokine release (Jaffre et al. 2009). Collectively, these results revealed that convergent action of norepinephrine, AngII, and serotonin via interactions between AT1 and 5-HT2B receptors coexpressed by noncardiomyocytes are limiting key events in cardiac hypertrophy.
Valvular Heart Disease
Carcinoid heart disease occurs in over 65% of patients with the carcinoid syndrome and is characterized by fibrous thickening of cardiac valves, leading to heart failure for review (Roth 2007). Correlation of high plasma serotonin levels with valvular abnormalities detected by cardiac catheterization and echocardiography has been reported. Thus, serotonin overproduction has been proposed to be responsible for cardiac valvular disease in patients with carcinoid tumors (Robiolio et al. 1995). The similarity to lesions in carcinoid heart disease and in methysergide-associated valvular disease suggested direct stimulation of myofibroblast growth by serotonin agonism (Hendrikx et al. 1996). The occurrence of fenfluramine-associated valvular heart disease has raised concerns that other serotonergic medications might also increase the risk of developing valvular heart disease (Connolly et al. 1997). The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) and its N-demethylated metabolite 3,4-methylenedioxyamphetamine (MDA) each preferentially bind to and activate human recombinant 5-HT2B receptors and like fenfluramine and its N-deethylated metabolite norfenfluramine elicit mitogenic responses in human valvular interstitial cells via activation of 5-HT2B receptors (Setola et al. 2003). Based on strikingly similar echocardiographic and histopathological features, it is now considered that ergot-derived dopamine agonists may cause a valvular heart disease nearly identical to that seen in patients with carcinoid syndrome (Horvath et al. 2004): population studies of patients with Parkinson’s disease compared with non-parkinsonian controls have reported that pergolide and cabergoline have a similar risk of inducing fibrotic changes in cardiac valve leaflets. Pergolide and cabergoline have high affinity for the 5-HT2B serotonin receptors (Antonini and Poewe 2007; Roth 2007). A simultaneous mitral bioprosthesis hypertrophic scaring and native aortic valve stenosis was recently reported during benfluorex therapy in a 40-year-old woman. The bioprosthesis and aortic valves exhibited similar histopathological lesions. Thickening and plaque deposits made by smooth muscle α-actin- and vimentin-positive cells in a glycosaminoglycan matrix were observed supporting that 5-HT2B receptors activated by norfenfluramine (also metabolite of benfluorex) trigger the development of drug-induced heart disease (Ayme-Dietrich et al. 2012). 5-HT2B and 5-HT2A receptor transcripts are easily detected in heart valves, while no 5-HT2C receptor transcript is detectable. Preferential stimulation of valvular 5-HT2B receptors (with or without accompanying 5-HT2A receptor activation) may contribute to valvular fibroplasia in humans (Fitzgerald et al. 2000). Mitral valve regurgitation has been associated with increased mRNA expression of valvular 5-HT2B receptors and SERT in pigs (Cremer et al. 2015b). Canine myxomatous mitral valve disease was associated with higher expression of 5-HT2B receptors in mitral valve (Cremer et al. 2015a).
These findings suggest that 5-HT2B signaling links vascular damage and platelet activation to tissue remodeling and identifies 5-HT2B as a novel therapeutic target to treat valvular heart diseases.
Serotonin released from mast cells or platelets in peripheral tissues is one important inflammatory mediators of pain. The involvement of serotonin in pain is complex, because it could inhibit or facilitate nociceptive transmission, reflecting the presence of multiple serotonin receptor subtypes on peripheral and central nociceptors. An acute injection of serotonin or the 5-HT2 agonist α-methyl-serotonin in hindpaws of mice induces significant hyperalgesia to mechanical stimuli, which can be inhibited by the 5-HT2B/2C antagonist SB206553 that also blocks serotonin-induced transient [Ca2+] signaling in DRG neurons. Thus, 5-HT2B receptors are involved in mechanical hyperalgesia in mice (Lin et al. 2011). The Gq/11-PLCCβ/protein kinase Cε (PKCε) pathway mediated by 5-HT2B receptor is involved in 5-HT-induced mechanical hyperalgesia in mice. Administration of a transient receptor potential vanilloid 1 (TRPV1) antagonist inhibits the 5-HT-induced mechanical hyperalgesia. 5-HT injection enhanced 5-HT- and capsaicin-evoked calcium signals that are inhibited by a 5-HT2B/2C antagonist and PKCε blocker. Thus, 5-HT2B mediates 5-HT-induced mechanical hyperalgesia by regulating TRPV1 function (Su et al. 2016).
Serotonin released from descending pain modulation pathways to the dorsal horn is also crucial to spinal nociception processing. Local peripheral ipsilateral but not contralateral injection of RS127445 significantly prevented 1% formalin-induced flinching behavior. Moreover, local peripheral ipsilateral but not contralateral injection of the 5-HT2 receptor agonist DOI augmented 0.5% formalin-induced nociceptive behavior. The local pronociceptive effect of DOI was significantly prevented by the local injection of RS127445. Moreover, intrathecal injection of RS127445 also prevented 1% formalin-induced nociceptive behavior. In contrast, spinal injection of DOI significantly increased flinching behavior induced by 0.5% formalin. The spinal pronociceptive effect of DOI was prevented by the intrathecal injection of RS127445 (Cervantes-Durán et al. 2012). These results suggest that 5-HT2B receptors play a pronociceptive role in peripheral as well as spinal sites in the rat formalin test.
In rats subjected to spinal nerve ligation surgery, the preferential 5-HT2B agonist BW723C86 was reported to enhance evoked potentials, while the 5-HT2B receptor-antagonist SB204741 depressed evoked potentials. Spinal hyperexcitation promoted by 5-HT2B receptors has been proposed as a pathogenic pathway contributing to pain (Aira et al. 2010). Plasticity of spinal serotonergic neurotransmission selectively reduces spinal μ-opioid receptor mechanisms via 5-HT2B receptors increased expression in dorsal horn neurons containing μ-opioid receptor (Aira et al. 2012). The involvement of glutamate receptors in dorsal neuron hyperexcitation can also be promoted by 5-HT2B receptor after spinal nerve ligation. Augmentation of C-fiber-evoked potentials by spinal superfusion with 5-HT2B receptor agonist BW723C86 in nerve-ligated rats is impeded by coadministration of NMDA receptor antagonist. Evoked potentials are increased by NMDA receptor agonist in nerve-injured rats, irrespective of simultaneous 5-HT2B receptor blockade by SB204741. In uninjured rats, NMDA receptor agonist enhanced evoked potentials in the presence of BW723C86 but not if administered alone. Blockade of 5-HT2B receptors with selective antagonist SB204741 after spinal nerve ligation bilaterally decreases phosphorylation of NMDA receptor subunit NR1 (pNR1) in synaptic fraction. Delivery of SB204741 bilaterally attenuates thermal and mechanical allodynia occurring after spinal nerve ligation, at early time period, day 2 postinjury. These findings suggest that transient activation of the PKCγ/NMDAR pathway is critically involved in 5-HT2B receptor-mediated facilitation in the spinal nerve ligation model (Aira et al. 2013). The adaptor protein NADH dehydrogenase subunit 2 (ND2) is involved in NR1-phosphorylation and spinal hyperexcitability secondary to peripheral nerve injury. Spinal nerve ligation is followed by increased co-localization of ND2 with pNR1. C fiber-evoked dorsal horn field potentials are increased 60 min after spinal nerve ligation by superfusion with NMDA agonist. This increased postsynaptic upregulation of ND2/pNR1 can be prevented by prior administration of selective 5-HT2B antagonist SB204741 (Aira et al. 2014). These results provide evidence that NMDAR phosphorylation is instrumental in coupling 5-HT2B receptor-mediated input to NMDAR-expressing synapses in spinal hyperexcitation involved in pain.
Serotonin has also been involved in a rat model of neuropathic pain evoked by chronic constriction injury (CCI) of the sciatic nerve. The 5-HT2B receptor activation has been reported to both prevent and reduce CCI-induced allodynia at 3 weeks postinjury. Intrathecal administration of the 5-HT2B receptor agonist BW723C86 significantly attenuated established mechanical and cold allodynia; this effect was prevented by co-injection of RS127445. A single application of BW723C86 on the sciatic nerve concomitantly to CCI dose-dependently prevented mechanical allodynia and significantly reduced cold allodynia 17 days after CCI. This behavioral effect was accompanied with a marked decrease in macrophage infiltration into the sciatic nerve and, in the DRG, with an attenuated abnormal expression of several markers associated with local neuroinflammation and neuropathic pain. CCI resulted in a marked upregulation of 5-HT2B receptor expression in sciatic nerve and DRG. In the latter structure, it is biphasic, consisting of a transient early increase (23-fold), 2 days after surgery and before neuropathic pain emergence, followed by a steady (fivefold) increase, that remains constant until pain disappeared. In DRG and sciatic nerve, 5-HT2B receptors are immunolocalized on sensory neurons and infiltrating macrophages (Urtikova et al. 2012).
A role for serotonin in migraines has been supported by changes in circulating levels of serotonin and its metabolites during the phases of a migraine attack. A migraine headache is thought to be transmitted by the trigeminal nerve from the meninges and their associated blood vessels. Correlation of the receptor affinities with the potencies used in migraine prophylaxis showed significant correlations for the 5-HT2B receptor. Various human meningeal tissues express 5-HT2B mRNAs (Schmuck et al. 1996). The 5-HT2B receptor can activate the release of NO and induce relaxation of the cerebral arteries and the jugular vein. 5-HT2B receptors located in endothelial cells of meningeal blood vessels may trigger migraine headache through the formation of NO, which results in the dilation of cerebral blood vessels and the concomitant activation of sensory trigeminovascular afferents, thus initiating the manifestation of head pain (Schmuck et al. 1996; Johnson et al. 2003). In addition, a recent genetic study identified 5-HT2B receptors as a susceptibility gene to migraine (Corominas et al. 2010). Endothelial 5-HT2B receptors may thus trigger dilation of meningeal blood vessels, which by activating sensory trigeminovascular afferents induces head pain.
5-HT2B receptors are also involved in signaling from the colon in rats in which there is visceral hypersensitivity. Oral administration of RS127445 significantly inhibits visceral hypersensitivity provoked by restraint stress without significant effect on the visceral nociceptive threshold of naïve rats (Ohashi-Doi et al. 2009). Moreover, when administered intracerebroventricularly RS127445 also decreases the number of pain behaviors during noxious colorectal distension. A selective 5-HT2B receptor antagonist has thus been proposed to have therapeutic potential for the treatment of gut disorders characterized by visceral hypersensitivity (O’Mahony et al. 2010). The 5-HT2B receptor appears thus to be involved in regulating sensory pathways only under hyperalgesic conditions, suggesting the possible utility of 5-HT2B receptor antagonism in reducing visceral hypersensitivity in patients with irritable bowel syndrome.
It thus appears that 5-HT2B receptor involvement in pain takes place at various sites and time periods, early events being more pronociceptive while at later this receptor contribution may be more antinociceptive, although this may vary according to the animal models.
The Htr 2B mRNA expression, which was undetectable in anaplastic osteoblasts, appears in differentiated and matured osteoblasts (Bliziotes et al. 2001; Westbroek et al. 2001). The differentiation and maturation of osteoblasts might thus be regulated by the activation of the 5-HT2B receptor (Hirai et al. 2009). The HTR 2A was found expressed only in osteoblasts, whereas HTR 2B expression increased from precursor to mature osteoclasts (Hodge et al. 2013). Of interest, Htr 2B −/− female mice displayed reduced bone density that was significant from the age of 4 months and intensifies by 12 and 18 months. This result confirmed that osteopenia was due to reduced bone formation because (i) the alkaline phosphatase-positive colony-forming unit capacity of bone marrow precursors was markedly reduced in Htr 2B −/− mice from 4 to 12 months of age, (ii) ex vivo primary osteoblasts from Htr 2B −/− mice exhibited reduced proliferation and delayed differentiation, and (iii) calcium incorporation was markedly reduced in osteoblasts after 5-HT2B receptor depletion (produced genetically or by pharmacological inactivation) (Collet et al. 2008). Using the osteoprogenitor cell line C1, blockade of 5-HT2B receptor intrinsic activity was reported to affect the efficiency of mineralization by decreasing calcium incorporation. Optimal bone matrix mineralization involves both NO and PLA2 signaling pathways, and the 5-HT2B receptor promotes prostaglandin E2 production through cyclooxygenase (COX) activation. When C1 osteoblasts undergo conversion into osteocyte-like cells, COX activity was quenched. The 5-HT2B receptor contributed in an autocrine manner to osteogenic differentiation (Locker et al. 2006). A functional link between the 5-HT2B receptor and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) was established. Agonist stimulation of the receptor increased TNAP activity during the initial mineralization phase. Indeed, inhibition of 5-HT2B receptor intrinsic activity prevented TNAP activation. In contrast, agonist stimulation of the receptor further increased TNAP activity during the initial mineralization phase. Previous observations indicated that the 5-HT2B receptor coupled to PLA2 pathway and prostaglandin production at the beginning of mineral deposition. The 5-HT2B receptor also controlled leukotriene synthesis via PLA2 at the terminal stages of differentiation. These two 5-HT2B receptor-dependent eicosanoid productions delineate distinct time-windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes were shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from Htr 2B −/− mice were shown to exhibit defects in TNAP activity (Baudry et al. 2010). Brain serotonin was proposed to favor indirectly bone mass accrual following activation of 5-HT2C receptors on ventromedial hypothalamic neurons and 5-HT2B receptors on arcuate neurons (Yadav et al. 2009). Compared to control osteoblasts, the lack of 5-HT2B receptors was associated with a 10-fold overproduction of prostacyclin (PGI2). Also, a specific prostacyclin synthase inhibitor (U51605) rescued totally osteoblast aggregation and matrix mineralization in Htr 2B −/− osteoblasts without having any effect on WT osteoblasts. Prostacyclin is the endogenous ligand of PPAR-β/δ, and its inhibition in Htr 2B −/− cells rescued totally the alkaline phosphatase and osteopontin mRNA levels, cell-cell adhesion, and matrix mineralization. The absence of 5-HT2B receptors leads to the overproduction of prostacyclin, inducing reduced osteoblast differentiation due to PPAR-β/δ -dependent target regulation and defective cell-cell adhesion and matrix mineralization (Chabbi-Achengli et al. 2013). The 5-HT2B receptor contributes thus in an autocrine manner to osteogenic differentiation.
5-HT2B receptor expression was observed in spontaneous human carcinoid tumors, along with coupling to p21ras activation (Launay et al. 1996). The tumor proliferative activity of small intestinal neuroendocrine tumors (including cell growth and the development of desmoplasia) is associated with the particular microenvironment in the peritoneum, and tumor cells support this necessary milieu through the secretion of profibrotic/ angiogenetic factors (Svejda et al. 2010).
Increased serotonin biosynthetic capacity accompanied by multiple changes in serotonin receptor expression and signaling favor malignant progression of human breast cancer cells. Among them, expression levels for 5-HT2B receptors was found increased in breast cancers and the HTR 2B mRNA is also expressed in untransformed human mammary epithelium (Pai et al. 2009). HTR 2B mRNA expression was found lower in basal tumors estrogen receptor (ER) negative, compared with luminal tumors, which are most commonly ER positive. HTR 2B mRNA was elevated in carcinomas, increased with tumor stage, and concomitantly was higher in lymph node-positive tumors as compared to node-negative tumors. This observation was supported by a study, showing that c-Myc transformation induced an increase in HTR 2B expression (Pai et al. 2009). In human breast cancer, correlation analysis revealed a significant correlation of HTR 2B with estrogen receptor-α (ER-α) (Kopparapu et al. 2013).
Uveal (ocular) melanoma is an aggressive cancer that often forms undetectable micrometastases before diagnosis of the primary tumor. High increases in HTR 2B mRNA transcript levels were found in all uveal melanomas with monosomy 3 compared with low expression in all tumors with disomy 3. As monosomy 3 is associated with metastatic disease, HTR 2B expression has been proposed as a marker to identify patients with poor prognosis (Tschentscher et al. 2003). The 5-HT2B receptor signals through the heterotrimeric GTPase, GNAQ, which is mutated in half of uveal melanomas (Onken et al. 2010). The 5-HT2B receptor is among the genes, which show the highest overexpression in class 2 uveal melanoma (van Gils et al. 2008). A PCR-based 15-gene assay comprising 12 discriminating genes including HTR 2B are now part of a prognostic assay, which provides an important addition to the armamentarium for managing patients with uveal melanoma (Onken et al. 2010). These genes, including HTR 2B , provide candidates for distinguishing whether uveal melanomas contain liver metastases and thus aid in the diagnosis and prevention of uveal melanoma liver metastases, based on their different features (Zhang et al. 2014).
Prostate cancer is the most commonly diagnosed noncutaneous cancer in men. Despite this fact, many of the genetic changes that coincide with prostate cancer progression remain enigmatic. The 5-HT2B receptor was found to be upregulated in tumors relative to benign glands (Magee et al. 2001). Overexpression of receptors to neuroendocrine cell products has been suggested to contribute to development of hormone-refractory prostate cancer. Immunostaining for the 5-HT2B receptor was observed in low-grade and high-grade tumors, prostatic intraepithelial neoplastic and benign prostatic hyperplasia cells, and in vascular endothelial cells. Antagonists for the 5-HT2B receptor inhibited proliferation of prostate cancer cells in a dose-dependent manner (Dizeyi et al. 2005).
By contrast, gene expression profiles of adrenocortical tumors identified underexpression of HTR 2B mRNA as a marker of malignant adrenocortical carcinoma (Fernandez-Ranvier et al. 2008). Analysis of biomarkers of malignancy of adrenocortical cancers in the meta-analysis has revealed that the combination of overexpressed anillin (ANLN) and underexpressed HTR 2B mRNA appeared to be the best predictor of malignancy (Zsippai et al. 2011). Chronic adrenal stimulation by glucose-dependent insulinotropic peptide in macronodular adrenal hyperplasia was shown to lead to the significant induction of the GPR54, HTR 2B , GPR4, and endothelial differentiation sphingolipid receptor EDG8 (Lampron et al. 2006).
Serotonin has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells. Among 64 genes for which mRNA expression levels differed between non-hepatitis B, non-hepatitis C compared to hepatitis C-type HCC, the most affected was found HTR 2B (Iizuka et al. 2004). The function of serotonin as a survival factor of HCC cells was recently demonstrated: Activation of the 5-HT2B receptor leads to sustained phosphorylation of two downstream targets of mTOR, p70S6K and 4E–BP1, thereby facilitating survival and inhibiting autophagy. Inhibiting the 5-HT2B receptor reduced cancer cell growth in vitro and in vivo. The presence of 5-HT2B receptors in HCC and the activation of autophagy-related mechanisms demonstrate novel insights of serotonin in cancer biology (Soll et al. 2010). The 5-HT1B and 5-HT2B receptors were found expressed, respectively, in 32% and 35% of the patients with hepatocellular cancer. Both receptors were associated with an increased proliferation index (Soll et al. 2012). The 5-HT2B receptor mediates serotonin-induced proliferation in the serum-deprived HCC Huh7 cells. Additionally, inhibition of 5-HT2B receptor in Huh7 cells using SB204741 significantly decreased the expression of FOXO3a (Liang et al. 2013). In vitro data suggest that 5-HT increased total β-catenin and active β-catenin and decreased phosphorylated β-catenin protein levels in serum-deprived Huh-7 and HepG2 cells compared to control cells under serum-free medium without 5-HT. Activation of Wnt/β-catenin signaling was evidenced by increased expression of β-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1), and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, 5-HT disrupted Axin1/β-catenin interaction, a critical step in β-catenin phosphorylation (Fatima et al. 2016).
In tumor-infiltrating macrophages, serotonin does not enhance colon cancer tumor cell proliferation but may act as a regulator of angiogenesis by reducing the expression of MMP-12, entailing lower levels of angiostatin – an endogenous inhibitor of angiogenesis (Nocito et al. 2008). Serotonin can stimulate the phosphorylation of ERK1/2 in bovine endothelial cells, and the 5-HT2B receptor was reported to play a role in the activation of eNOS in human endothelial cells. In SB204741-treated mice, the selective blockade of the 5-HT2B receptor resulted in the reduction of tumor angiogenesis and growth through the inhibition effect of ERK1/2 and eNOS (Asada et al. 2009). Therefore the possibility that 5-HT2B receptors participate in tumor angiogenesis is a likely possibility that remains to be evaluated in other tumors subtypes.
Although expressed quite widely but at rather low levels, 5-HT2B receptors have been reported to play an important role at cardiac, pulmonary, and central levels. Therapeutic interest concerning this receptor subtype has been delayed due to the lack of selective tools, radioligand, antibodies etc. However, selective antagonists at 5-HT2B receptors have gained much attention as new targets in therapeutics: 5-HT2B receptor antagonists are under active investigation for pulmonary hypertension and carcinoid tumors, since they produced encouraging results in animal models, and promising human trials are underway; 5-HT2B receptor may represent a new therapeutic target to reduce cardiac hypertrophy and hypertrophy-induced heart failure as well as valvulopathies. If the safety and efficacy of 5-HT2B receptor antagonists can be confirmed, application of this kind of therapy to human study may be warranted. These receptors have also been recognized as off-targets, because their agonist plays a significant role in the pathogenesis of valvulopathy and pulmonary hypertension.
- Asada M, Ebihara S, Yamanda S, Niu K, Okazaki T, Sora I, Arai H. Depletion of serotonin and selective inhibition of 2B receptor suppressed tumor angiogenesis by inhibiting endothelial nitric oxide synthase and extracellular signal-regulated kinase 1/2 phosphorylation. Neoplasia. 2009;11(4):408–17.PubMedPubMedCentralCrossRefGoogle Scholar
- Baudry A, Bitard J, Mouillet-Richard S, Locker M, Poliard A, Launay J-M, Kellermann O. The serotonergic 5-HT2B receptor controls tissue non-specific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C. J Biol Chem. 2010;285(34):26066–73.PubMedPubMedCentralCrossRefGoogle Scholar
- Cohen ML, Schenck KW, Mabry TE, Nelson DL, JE A. LY272015, a potent, selective and orally active 5-HT2B receptor antagonist. J Serv Res. 1996;3:131–44.Google Scholar
- Cussac D, Newman-Tancredi A, Quentric Y, Carpentier N, Poissonnet G, Parmentier JG, Millan MJ. Characterization of phospholipase C activity at h5-HT2C compared with h5-HT2B receptors: influence of novel ligands upon membrane-bound levels of [3H]phosphatidylinositols. Naunyn Schmiedeberg's Arch Pharmacol. 2002;365(3):242–52.CrossRefGoogle Scholar
- Cussac D, Boutet-Robinet E, Ailhaud MC, Newman-Tancredi A, Martel JC, Danty N, Rauly-Lestienne I. Agonist-directed trafficking of signalling at serotonin 5-HT2A, 5-HT2B and 5-HT2C-VSV receptors mediated Gq/11 activation and calcium mobilisation in CHO cells. Eur J Pharmacol. 2008;594(1–3):32–8.PubMedCrossRefGoogle Scholar
- Jaffré F, Callebert J, Sarre A, Etienne N, Nebigil CG, Launay JM, Monassier L. Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts. Circulation. 2004;110:969–74.PubMedCrossRefGoogle Scholar
- Kiss B, Horváth A, Némethy Z, Schmidt E, Laszlovszky I, Bugovics G, Szombathelyi Z. Cariprazine (RGH-188), a dopamine D(3) receptor-preferring, D(3)/D(2) dopamine receptor antagonist-partial agonist antipsychotic candidate: in vitro and neurochemical profile. J Pharmacol Exp Ther. 2010;333(1):328–40.PubMedCrossRefGoogle Scholar
- Kolodziejczak M, Bechade C, Gervasi N, Irinopoulou T, Banas SM, Cordier C, Maroteaux L. Serotonin modulates developmental microglia via 5-HT2B receptors: potential implication during synaptic refinement of retinogeniculate projections. ACS Chem Neurosci. 2015;6(7):1219–30.PubMedCrossRefGoogle Scholar
- Lampron A, Bourdeau I, Hamet P, Tremblay J, Lacroix A. Whole genome expression profiling of glucose-dependent insulinotropic peptide (GIP)- and adrenocorticotropin-dependent adrenal hyperplasias reveals novel targets for the study of GIP-dependent Cushing’s syndrome. J Clin Endocrinol Metab. 2006;91(9):3611–8.PubMedCrossRefGoogle Scholar
- Millan MJ, Gobert A, Lejeune F, Dekeyne A, Newman-Tancredi A, Pasteau V, Cussac D. The novel melatonin agonist agomelatine (S20098) is an antagonist at 5-hydroxytryptamine2C receptors, blockade of which enhances the activity of frontocortical dopaminergic and adrenergic pathways. J Pharmacol Exp Ther. 2003;306(3):954–64.PubMedCrossRefGoogle Scholar
- Osredkar D, Krzan M. Expression of serotonin receptor subtypes in rat brain and astrocyte cell cultures: an age-and tissue-dependent process. Period Biol. 2009;111(1):129–35.Google Scholar
- Porvasnik SL, Germain S, Embury J, Gannon KS, Jacques V, Murray J, Al-Mousily F. PRX-08066, a novel 5-hydroxytryptamine receptor 2B antagonist, reduces monocrotaline-induced pulmonary arterial hypertension and right ventricular hypertrophy in rats. J Pharmacol Exp Ther. 2010;334(2):364–72.PubMedCrossRefGoogle Scholar
- Svejda B, Kidd M, Giovinazzo F, Eltawil K, Gustafsson BI, Pfragner R, Modlin IM. The 5-HT2B receptor plays a key regulatory role in both neuroendocrine tumor cell proliferation and the modulation of the fibroblast component of the neoplastic microenvironment. Cancer. 2010;116(12):2902–12.PubMedCrossRefGoogle Scholar
- Watts SW, Fink GD. 5-HT2B-receptor antagonist LY-272015 is antihypertensive in DOCA-salt- hypertensive rats. Am J Phys. 1999;276(3 Pt 2):H944–52.Google Scholar
- Yan L, Chen X, Talati M, Nunley BW, Gladson S, Blackwell T, Hamid R. Bone marrow-derived cells contribute to pathogenesis of pulmonary arterial hypertension. Am J Resp Crit Care Med. 2016;193(8):898–909.Google Scholar