Serotonin in pain and analgesia

The purpose of this article is to summarize recent findings on the role of serotonin in pain processing in the peripheral nervous system. Serotonin (5-hydroxtryptamine [5-HT]) is present in central and peripheral serotonergic neurons, it is released from platelets and mast cells after tissue injury, and it exerts algesic and analgesic effects depending on the site of action and the receptor subtype. After nerve injury, the 5-HT content in the lesioned nerve increases. 5-HT receptors of the 5-HT₃ and 5-HT_2A subtype are present on C-fibers. 5-HT, acting in combination with other inflammatory mediators, may ectopically excite and sensitize afferent nerve fibers, thus contributing to peripheral sensitization and hyperalgesia in inflammation and nerve injury.     

A paracrine signaling role for serotonin in rat taste buds: expression and localization of serotonin receptor subtypes

Recent advances in peripheral taste physiology now suggest that the classic linear view of information processing within the taste bud is inadequate and that paracrine processing, although undemonstrated, may be an essential feature of peripheral gustatory transduction. Taste receptor cells (TRCs) express multiple neurotransmitters of unknown function that could potentially participate in a paracrine role. Serotonin is expressed in a subset of TRCs with afferent synapses; additionally, TRCs respond physiologically to serotonin. This study explored the expression and cellular localization of serotonin receptor subtypes in TRCs as a possible route of paracrine communication. RT-PCR was performed on RNA extracted from rat posterior taste buds with 14 prime sets representing 5-HT(1) through 5-HT(7) receptor subtype families. Data suggest that 5-HT(1A) and 5-HT(3) receptors are expressed in taste buds. Immunocytochemistry with a 5-HT(1A)-specific antibody demonstrated that subsets of TRCs were immunopositive for 5-HT(1A). With the use of double-labeling, serotonin- and 5-HT(1A)-immunopositive cells were observed exclusively in nonoverlapping populations. On the other hand, 5-HT(3)-immunopositive taste receptor cells were not observed. This observation, combined with other data, suggests 5-HT(3) is expressed in postsynaptic neural elements within the bud. We hypothesize that 5-HT release from TRCs activates postsynaptic 5-HT(3) receptors on afferent nerve fibers and, via a paracrine route, inhibits neighboring TRCs via 5-HT(1A) receptors. The ole of the 5-HT(1A)-expressing TRC within the taste bud remains to be explored.     

Cell-to-Cell Communication in the Taste Bud: ATP and Acetylcholine as Primary Mediators

It was shown that physiological processes in taste buds (peripheral sensory gustatory organs in vertebrates) are realized with the involvement of several signal systems. In these structures, a number of “classical” neurotransmitters, including glutamate, serotonin, GABA, ATP, noradrenaline, and others, as well as receptors to these agents, were identified. The physiological roles of the above systems (separate ones and all as a whole) remain, however, far from final elucidation. We studied purinergic and cholinergic systems in the taste buds. Based on the data obtained in behavioral experiments using knockout animals, which indicated that ATP is an afferent neurotransmitter, we found stimulation-induced secretion of ATP by type-II cells. The release of ATP does not require the entry of external calcium and is mediated by ion channels permeable for ATP. The obtained data allowed us to explain the fact that classical synaptic structures are absent in the type-II cells. The type-I cells coat other elements including type-II cells; they provide formation of compartments in the intercellular space of the taste buds (this limits ATP diffusion). We showed that taste cells of just type I mostly generate calcium signals in response to the action of ATP and acetylcholine. These cell responses are generated with the involvement of metabotropic purine receptors (isoforms P2Y1, P2Y2, and P2Y4) and muscarinic receptors (isoforms M1, M3, and M5), respectively. Functioning of these receptors is combined with a phosphoinositide cascade, mobilization of intracellular Ca²⁺, and subsequent activation of calcium-activated Cl^– channels. It seems probable that purinergic and cholinergic signal systems in type-I cells are elements of negative feedback in the taste buds, which promote the process of adaptation to the action of gustatory stimuli.     

Evolutionary origins of taste buds: phylogenetic analysis of purinergic neurotransmission in epithelial chemosensors

Taste buds are gustatory endorgans which use an uncommon purinergic signalling system to transmit information to afferent gustatory nerve fibres. In mammals, ATP is a crucial neurotransmitter released by the taste cells to activate the afferent nerve fibres. Taste buds in mammals display a characteristic, highly specific ecto-ATPase (NTPDase2) activity, suggesting a role in inactivation of the neurotransmitter. The purpose of this study was to test whether the presence of markers of purinergic signalling characterize taste buds in anamniote vertebrates and to test whether similar purinergic systems are employed by other exteroceptive chemosensory systems. The species examined include several teleosts, elasmobranchs, lampreys and hagfish, the last of which lacks vertebrate-type taste buds. For comparison, Schreiner organs of hagfish and solitary chemosensory cells (SCCs) of teleosts, both of which are epidermal chemosensory end organs, were also examined because they might be evolutionarily related to taste buds. Ecto-ATPase activity was evident in elongate cells in all fish taste buds, including teleosts, elasmobranchs and lampreys. Neither SCCs nor Schreiner organs show specific ecto-ATPase activity, suggesting that purinergic signalling is not crucial in those systems as it is for taste buds. These findings suggest that the taste system did not originate from SCCs but arose independently in early vertebrates.     

5-HT2 Receptors Facilitate JC Polyomavirus Entry

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT_2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT_2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT₂ receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT₂ receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT₂ receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.     

The Human Serotonin 1A Receptor Expressed in Neuronal Cells: Toward a Native Environment for Neuronal Receptors

1. The serotonin_1A (5-HT_1A) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions.2. We report here the pharmacological and functional characterization of the human serotonin_1A receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma.3. Our results show that serotonin_1A receptors in HN2-5-HT_1AR cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT_1AR cells. Importantly, the serotonin_1A receptor in HN2-5-HT_1AR cells shows efficient downstream signalling by reducing cellular cyclic AMP levels.4. We conclude that serotonin_1A receptors expressed in HN2-5-HT_1AR cells represent a useful model system to study serotonin_1A receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

Genetic tracing of the gustatory neural pathway originating from Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae

Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate (CvP) and foliate papillae (FoP) located in the posterior region of the tongue, although not in fungiform papillae (FuP) or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in CvP and FoP, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in CvP and FoP. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal (GL) nerve bundles in the nodose/petrosal ganglion (NPG). WGA signals were also observed in a small population of neurons in the geniculate ganglion (GG). This result demonstrates the anatomical connection between taste receptor cells (TRCs) in the FoP and the chorda tympani (CT) nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract (NST). These findings demonstrate that the approximately 10?kb 5'-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from TRCs in the posterior region of the tongue.     

Phosphatidylinositol 4-phosphate 5-kinase Iγ_v6, a new splice variant found in rodents and humans

It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT₃) receptor to cause nausea and vomiting. In general, antagonist for 5-HT₃ receptor is used as anti-emetics during chemotherapy. However, we found that anti-cancer drug irinotecan itself inhibits 5-HT-gated current through the homomeric 5-HT_3A and heteromeric 5-HT_3AB receptor in a concentration-dependent manner. The inhibitory effect of irinotecan on 5-HT_3A receptor was more potent than that on 5-HT_3AB receptor. On the other hand, SN-38, a metabolite of irinotecan, had no effect on the responsiveness. Our findings suggest that irinotecan itself could have anti-emetic activities through inhibition of the 5-HT_3A and 5-HT_3AB receptor.     

Relationship between 5-HT2A receptor mRNA density and contractility in trachea and aorta from guinea pig and rat

The present studies document marked differences in contractile responsiveness to serotonin in trachea and aorta between guinea pig and rat. For example, the guinea pig trachea and rat aorta markedly contract in response to serotonin via activation of 5-HT_2A receptors. In contrast, the rat and guinea pig aorta only modestly contract to serotonin. The availability of 5-HT_2A receptor selective cDNA clones from brain of both guinea pig and rat permitted molecular probes to be designed and PCR amplification studies initiated to identify and quantify 5-HT_2A receptor specific mRNA in these tissues. For trachea, 3-fold higher concentrations of 5-HT_2A receptor specific mRNA were found in guinea pig relative to rat trachea. These data are consistent with the more profound contractile response to serotonin in guinea pig versus rat trachea and suggest that differences in tracheal contractility to serotonin correlate with the density of 5-HT_2A receptor mRNA. In contrast, although rat aorta contracted more dramatically to serotonin than guinea pig aorta, rat aorta possessed a similar concentration of 5-HT_2A receptor specific mRNA as compared to guinea pig aorta. Thus, for the aorta, differences in the concentration of 5-HT_2A receptor mRNA are not sufficient to explain the observed differences in contractility between tissues from guinea pig and rat. These studies documenting 5-HT_2A receptor mRNA in rat trachea and guinea pig aorta, two tissues that do not markedly contract in response to serotonin indicate that 5-HT_2A receptor mRNA although present, has not resulted in a receptor capable of mediating a contractile response in these tissues.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT_1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin_1A receptor tagged to enhanced yellow fluorescent protein (5-HT_1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT_1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT_1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT_1AR-EYFP provide new insights into the membrane organization of the 5-HT_1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.     

The 5-HT1A receptor: an overview of recent advances

Progress in the field of neuronal receptor research has accelerated during the last few years due to developments in pharmacology and molecular biology. This is particularly true in the case of the serotonin 5-HT_1A receptor. In 1983 the very selective, high affinity 5-HT_1A agonist 8-OH-DPAT was developed which allowed the pharmacology and distribution of the 5-HT_1A receptor in the central nervous system of the rat and man to be extensively characterized. By 1987, the gene encoding this receptor protein was cloned and sequenced, allowing not only elucidation of its structure, but also better insight into the nature of its coupling to transmembrane signal transduction systems. Thus in a short period of time considerable knowledge has accumulated on how serotonin exerts its functions in the central nervous system via the 5-HT_1A receptor. In the present review we will briefly discuss some of the latest developments regarding the 5-HT_1A receptor.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Spiro[pyrrolidine-3,3′-oxindoles] as 5-HT7 receptor ligands

Here we report the design and synthesis of spiro[pyrrolidine-3,3′-oxindole] derivatives representing a novel scaffold of 5-HT₇ receptor ligands. The synthesized analogues were validated as low nanomolar ligands showing selectivity in a panel of related serotonin receptor subtypes including 5-HT_1AR, 5-HT_2AR and 5-HT₆R.     

Identification of Three Residues Essential for 5-Hydroxytryptamine 2A-Metabotropic Glutamate 2 (5-HT2A·mGlu2) Receptor Heteromerization and Its Psychoactive Behavioral Function

Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT_2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-677^4.40, Ala-681^4.44, and Ala-685^4.48) leads to absence of 5-HT_2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT_2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT_2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer.     

5-ht2a Receptors in rat Sciatic Nerves and Schwann Cell Cultures

Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT_2A receptor (5-HT_2AR). In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT_2AR protein mRNA. We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT. Rat Schwann cells of a myelinating phenotype contained both 5-HT_2AR protein and mRNA. In the healthy adult rat sciatic nerve, 5-HT_2ARs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm. The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves. These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells.     

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling

The serotonin 5-HT₇ G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT₇. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT₇ GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT_1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure—affinity relationship (3D-QSAR) at the human 5-HT₇ receptor for comparison with similar studies at the highly homologous 5-HT_1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (K_i ≤ 1 nM) and stereoselectivity at 5-HT₇ + or 5-HT_1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT₇, and, several ligands with high selectivity (up to 40-fold) at the 5-HT_1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT₇ over 5-HT_1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT_1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT₇ vs. 5-HT_1A GPCRs, as may be required for successful clinical translation.