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.     

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.     

Influence of exercise on serotonergic neuromodulation in the brain

Summary. Implications of exercise on serotonergic neuromodulation in the brain have been investigated in two studies. Acute paroxetine (selective serotonin (5-HT) reuptake inhibitor) administration to endurance athletes, who performed a cycle ergometer test to exhaustion at moderate intensity, reduced time to exhaustion and post exercise cognitive performance in comparison to trials with placebo or BCAA administration. Furthermore, during a 3-week moderate endurance training of sedentary males basaline values of B_max of 5-HT transporters (5-HTT) and 5-HT_2A receptors (5-HT_2AR) on isolated platelet membranes increased while plasma prolactin (PRL) concentrations decreased as well as mood and physical efficiency improved. In contrast, after an excessive training program over four weeks, well-trained endurance athletes showed no change of B_max of 5-HTT, but a decline of 5-HT_2AR density and an increase in basal plasma PRL concentration. Mood was impaired and central fatigue increased. Thus, the impact of exercise on 5-HT neurotransmission may depend on training state of athletes and extent of exertion. The theoretical background of the implication of exercise and the effect of long lasting exhaustive exercise in athletes on mental and physical efficiency or central fatigue are evaluated. The significance of the primary disturbance of central neuromodulation and dysfunction of 5-HTT, 5-HT receptor subtypes and the phosphoinositol signal transduction as well as the limited modulation capacity of the 5-HT system in overstrain are also addressed. Received December 1, 1999 / Accepted February 1, 2000     

Alpha-ethyltryptamines as dual dopamine–serotonin releasers

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT_2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT_2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT_2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.     

Multiple Serotonin Receptors: Opportunities for New Treatments for Obesity?

Recent progress in the molecular pharmacology of 5-HT receptors and the development of selective ligands for various 5-HT receptor subtypes has advanced our understanding of the role of 5-HT mechanisms in the control of food intake and body weight The most intensively investigated 5-HT receptor subtypes have been the 5-HT_1A receptor, the 5-HT_1B receptor andthe5-HT_2C receptor. The overall pattern of results to date suggests that selective 5-HT_2C agonists may be novel anorectic drugs and prove useful in the treatment of obesity. However, a number of issues remain unresolved, particularly regarding potential side-effects, as the 5-HT_2C receptor agonist mCPP has been reported to induce anxiety and nausea in humans, actions that would clearly limit its therapeutic utility. In addition, the possible role of recently cloned 5-HT receptor subtypes such as 5-ht₅,5-ht₆ and 5-ht₇ remains unexplored and the development of selective ligands for these sites has the potential to lead to new treatments for obesity .     

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.     

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.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT_2A receptor. Active state of 5-HT_2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT_2A receptor active state model was constructed by homology modelling using active state β₂-adrenergic receptor (β₂-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.     

Synthesis and SAR of potent and selective tetrahydropyrazinoisoquinolinone 5-HT2C receptor agonists

The 5-HT_2C receptor has been implicated as a critical regulator of appetite. Small molecule activation of the 5-HT_2C receptor has been shown to affect food intake and regulate body weight gain in rodent models and more recently in human clinical trials. Therefore, 5-HT_2C is a well validated target for anti-obesity therapy. The synthesis and structure–activity relationships of a series of novel tetrahydropyrazinoisoquinolinone 5-HT_2C receptor agonists are presented. Several members of this series were identified as potent 5-HT_2C receptor agonists with high functional selectivity against the 5-HT_2A and 5-HT_2B receptors and reduced food intake in an acute rat feeding model upon oral dosing.     

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT_1,5A,7 receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT_1,7 receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT_1A receptors, as in Xenopus, but also 5-HT₇ receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT_1A and/or 5-HT₇ receptor antagonism, implicating 5-HT_1B/1D receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT_1A,7 and 5-HT_1B/1D receptors act as gain-setters of burst durations, whilst 5-HT_5A receptors are involved in the effects of bath-applied 5-HT on locomotion.     

Prefrontal Serotonergic Denervation Induces Increase in the Density of 5-HT<Subscript>2A</Subscript> Receptors in Adult Rat Prefrontal Cortex

The 5-HTergic system and particularly 5-HT_2A receptors have been involved in prefrontal cognitive functions, but the underlying mechanisms by which the serotonin (5-HT) system modulates these processes are still unclear. In this work, the effects of prefrontal 5-HTergic denervation on the density and expression levels of 5-HT_2A receptors were evaluated by immunohistochemical and molecular biology studies in the prefrontal cortex (PFC). The [³H]-Ketanserin binding study revealed an increase in the B_max, along with no change in the binding affinity (K_D) for 5-HT_2A receptors. The increase in PFC of 5-HT_2A receptor density in response to denervation was accompanied by increase in 5-HT_2A receptor mRNA and protein levels. This increase in the number of 5-HT_2A receptors may be interpreted as an adaptive plastic change, i.e., hypersensitivity; resulting from the selective pharmacological lesion of the raphe-proceeding 5-HTergic fibers to the PFC. Based on previous evidence, this could be strongly related to the abnormal expression of short-term memory.     

Design, synthesis, and pharmacological evaluation of azetedine and pyrrolidine derivatives as dual norepinephrine reuptake inhibitors and 5-HT(1A) partial agonists

Compounds with combined norepinephrine reuptake inhibitor (NRI) and serotonin 1A (5-HT_1A) partial agonist pharmacology may offer a new therapeutic approach for treating symptoms of neuropsychiatric disorders including ADHD, depression, and anxiety. Herein we describe the design and optimization of novel chemical matter that exhibits favorable dual NRI and 5-HT_1A partial agonist activity. Lead compounds in this series were found to be devoid of activity at the dopamine transporter and were shown to be brain penetrant with high receptor occupancy.     

Serotonin 5-HT3 receptors in the central nervous system

The 5-HT₃ receptor is a ligand-gated ion channel activated by serotonin (5-HT). Although originally identified in the peripheral nervous system, the 5-HT₃ receptor is also ubiquitously expressed in the central nervous system. Sites of expression include several brain stem nuclei and higher cortical areas such as the amygdala, hippocampus, and cortex. On the subcellular level, both presynaptic and postsynaptic 5-HT₃ receptors can be found. Presynaptic 5-HT₃ receptors are involved in mediating or modulating neurotransmitter release. Postsynaptic 5-HT₃ receptors are preferentially expressed on interneurons. In view of this specific expression pattern and of the well-established role of 5-HT as a neurotransmitter shaping development, we speculate that 5-HT₃ receptors play a role in the formation and function of cortical circuits.     

Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Molecular basis for cis-urocanic acid as a 5-HT2A receptor agonist

The underlying mechanisms of urocanic acid (UA) to induce immune suppression remain elusive until the recent finding that cis-UA acts via the serotonin, 5-hydroxytryptamine (5-HT) receptor subtype 5-HT_2A. In the present study, the interactions of cis-UA to 5-HT_2A receptor were explored and compared with those of 5-HT to the same receptor using computational docking. Similar binding modes were observed for cis-UA and 5-HT with 5-HT_2A receptor and the former possessed relatively higher binding affinity, which may account for cis-UA being a serotonin receptor agonist. Moreover, the molecular basis for the distinct binding affinities between the trans- and cis-UA with 5-HT_2A receptor was also provided.     

Decreased 5-HT<Subscript>1A</Subscript> Receptor Gene Expression and 5-HT<Subscript>1A</Subscript> Receptor Protein in the Cerebral Cortex and Brain Stem during Pancreatic Regeneration in Rats

The present study was to investigate the role of central 5-HT and 5-HT_1A receptor binding and gene expression in a rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content and 5-HT_1A receptor gene expression in the cerebral cortex (CC) and brain stem (BS) of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content significantly increased in the CC (P < 0.01) and BS (P < 0.05) of 72 h pancreatectomised rats. Sympathetic activity was decreased as indicated by the significantly decreased norepinephrine (NE) and epinephrine (EPI) level (P < 0.001 and P < 0.05) in the plasma of 72 h pancreatectomised rats. 5-HT_1A receptor density and affinity was decreased in the CC (P < 0.01) and BS (P < 0.01). These changes correlated with a diminished 5-HT_1Areceptor mRNA expression in the brain regions studied. Our results suggest that the brain 5-HT through 5-HT_1A receptor has a functional role in the pancreatic regeneration through the sympathetic regulation.     

Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: Multiple regulatory pathways control serotonergic neuronal differentiation

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV 40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotrophic hormone peptide ACTH_4–10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH_4–10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH_4–10 is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca²⁺ -channel blockers nifedipine and ω-conotoxin. ACTH_4–10 treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT_1A receptor is increased threefold by ACTH_4–10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH_4–10 treatment and membrane depolarization does not increase expression of the 5-HT_1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells. Considered with previous results, these data indicate that multiple effectors, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation. © 1995 John Wiley & Sons, Inc.     

A Naturally Occurring Amino Acid Substitution of the Human Serotonin 5-HT2A Receptor Influences Amplitude and Timing of Intracellular Calcium Mobilization

Abstract: Recently, two naturally occurring amino acid substitutions were identified in the C-terminal region of the serotonin 5-HT_2A receptor. One of these, His ⁴⁵²Tyr, has a rarer allele Tyr frequency of 9%. If ⁴⁵²Tyr alters 5-HT_2A function, it would thus be a candidate allele for human neurobehavioral variation. The present study was designed to evaluate the potential influence of the ⁴⁵²His and ⁴⁵²Tyr alleles on cellular 5-HT_2A functions. Platelet 5-HT_2A binding and 5-HT-induced Ca²⁺ response were compared in eight ⁴⁵²His/⁴⁵²His homozygous and eight ⁴⁵²His/⁴⁵²Tyr heterozygous individuals matched for sex, age, and diagnosis (all were patients with seasonal affective disorder). There was no difference in 5-HT_2A binding measured using ¹²⁵I-lysergic acid diethylamide. Nor were levels of G-protein subunits or PKC α, δ, ε, or ζ significantly altered. However, when Ca²⁺ response was stimulated by 2, 5, 10, or 25 µM 5-HT, significant differences were found. In ⁴⁵²His/⁴⁵²Tyr heterozygotes, ⁴⁵²Tyr was associated with both smaller peak amplitude in Ca²⁺ mobilization and a different time course of response, with slower peak latency and longer half-time in ⁴⁵²His/⁴⁵²Tyr heterozygotes compared with ⁴⁵²His/⁴⁵²His homozygotes. The overall difference in the response of the 5-HT_2A receptor in individuals with ⁴⁵²Tyr was a blunting of the shape of the Ca²⁺ mobilization peak. The data reported here suggest that the primary sequence of this intracellular domain is important in function of the receptor and that the ⁴⁵²His and ⁴⁵²Tyr 5-HT_2A alleles should be carefully evaluated for effects on human neurobehavioral variation.