Serotonin responsiveness through 5-HT2A and 5-HT4 receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle

Cardiac ventricular responsiveness to serotonin appears in rat postinfarction congestive heart failure (CHF), mainly mediated by 5-HT₄ receptors in chronic dilated CHF and 5-HT_2A receptors in acute CHF. To differentiate between the effects of left ventricular (LV) hypertrophy and failure on 5-HT_2A- and 5-HT₄-mediated inotropic serotonin response, male Wistar rats with increasing LV hypertrophy (AB1-3) and failure (ABHF) 6 weeks after banding of the ascending aorta were screened for contractile function in vivo (echocardiography) and ex vivo in LV papillary muscles, and mRNA expression level determined by RT-PCR. Both AB1-3 and ABHF displayed LV hypertrophy and remodelling. In ABHF, systolic LV and left atrial diameter increased and cardiac output decreased compared to AB3. Serotonin induced a positive inotropic response (PIR) in papillary muscles correlated with the degree of hypertrophy reaching a maximum in ABHF. Both 5-HT_2A and 5-HT₄ receptors contributed to the PIR. The 5-HT_2A contribution increased with increasing hypertrophy, and the 5-HT₄ contribution increased upon transition to heart failure. No 5-HT_2B-mediated PIR was observed, consistent with increased 5-HT_2B mRNA only in non-cardiomyocytes. The 5-HT_2A, 5-HT_2B and 5-HT₄ mRNA levels increased in AB1-3 and increased further in ABHF compared to AB3, but did not correlate with degree of hypertrophy. 5-HT_2A mRNA was also increased in LV of terminally failing human hearts. In conclusion, functional 5-HT_2A and 5-HT₄ receptors are differentially induced in LV hypertrophy and failure. While the 5-HT_2A-mediated PIR is linearly correlated with the degree of hypertrophy, the 5-HT₄-mediated PIR seems to increase with LV dilatation, as also seen in postinfarction CHF.     

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo

Objective

Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT₇ receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation.

Methods

Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2–4 rats/sample) to evaluate 5-HT₇ receptor expression.

Results

Topical 5-HT (1–10 nmols) or the 5-HT_1/7 receptor agonist, 5-carboxamidotryptamine (10–30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT₇ receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT₇ receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT₇ receptors.

Conclusions

5-HT₇ receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.     

Effects of Maternal Ethanol Consumption and Buspirone Treatment on 5-HT1A and 5-HT2A Receptors in Offspring

In utero ethanol exposure results in a decreased concentration of serotonin (5-HT) in brain regions containing the cell bodies of 5-HT neurons and their cortical projections. The concentration of 5-HT reuptake sites is also reduced in several brain areas. The present study extended prior work by evaluating the effects of chronic maternal ethanol consumption and maternal buspirone treatment on 5-HT_1A and 5-HT_2A receptors in multiple brain areas of offspring. Receptors were quantitated early in postnatal development and at an age when the 5-HT networks are normally well-established. Because fetal 5-HT functions as an essential neurotrophic factor, these studies also determined whether treatment of pregnant rats with buspirone, a 5-HT_1A agonist, could overcome the effects of the fetal 5-HT deficit and prevent ethanol-associated receptor abnormalities. The results demonstrated that in utero ethanol exposure significantly alters the binding of 0.1 nM [³H]-8-hydroxy-dipropylaminotetralin to 5-HT_1A receptors in developing animals. Ethanol impaired the development of 5-HT_1A receptors in the frontal cortex, parietal cortex, and lateral septum; these receptors did not undergo the normal developmental increase between postnatal days 19 and 35. The dentate gyms was also sensitive to the effects of in utero ethanol exposure. 5-HT_1A receptors were increased in this region at 19 days. Maternal buspirone treatment prevented the ethanol-associated abnormalities in 5-HT_1A receptors in the dentate gyms, frontal cortex, and lateral septum. Neither maternal ethanol consumption nor buspirone treatment altered the binding of 2 nM [³H]ketanserin to 5-HT_2A receptors in the ventral dentate gyrus, dorsal raphe, parietal and frontal cortexes, striatum, substantia nigra, or nucleus accumbens.     

Decrease in Gastric Sensitivity to Distension by 5-HT1A Receptor Agonists in Rats

Using an in vivo model for evaluation of gastricsensitivity in awake rats, we aimed to determine whether5-hydroxytryptamine 1A (5-HT_1A) agonistsmodify pain threshold and gastric compliancespecifically through 5-HT_1A receptors. Isobaricgastric distensions were performed with a barostat usingsteps of 5 mm Hg in male rats equipped with a gastricballoon and electrodes implanted in the neck muscles.Gastric distension at 15 or 20 mm Hg induced a typicalposture associated with contractions of the neckmuscles. Rats received drugs 30 min before gastricdistension. The 5-HT receptor agonist8-hydroxy-2-(di-n-propylamino)tetra1A lin (8-OH-DPAT),administered intraperitoneally (0.5 mg/kg) increasedgastric pain threshold and gastric tone. These effectswere reproduced when administered centrally (0.05 mg/kg) and blocked by intracerebroventricularadministration of the 5-HT_1A antagonist WAY100635. Flesinoxan (4 mg/kg, intraperitoneally), another5-HT_1A agonist reproduced the effects of8-OH-DPAT on pain threshold and gastric tone and the alpha-receptorantagonist yohimbine did not modify the action of8-OH-DPAT. Our results indicate that activation of5-HT_1A receptors at the level of the centralnervous system increases gastric tone and decreases gastric sensitivityto distension.     

Functional 5-HT receptor subtypes in the isolated canine common carotid artery

Summary The vascular responses to 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT, a selective 5-HT₁-like receptor agonist), alphamethyl-5-HT (-M-5-HT, a relatively selective 5-HT₂ receptor agonist), noradrenaline (NA), and KCl were examined in isolated, cannulated, and perfused canine common carotid arterial preparations. They caused strong vasoconstrictions. The rank order of vasoconstrictive potency was 5-HT > -M-5-HT NA > 5-CT >> KCl. The 5-HT-induced vasoconstriction was significantly depressed by methysergide (a 5-HT₁ and 5-HT₂ receptor antagonist), ketanserin (a selective 5-HT₂ receptor antagonist), and spiperone (a selective 5-HT₂ receptor antagonist). The 5-CT- and -M-5-HT-induced vasoconstrictions were also significantly inhibited by methysergide, spiperone, and ketanserin. The NA-induced vasoconstriction was readily inhibited by bunazosin (an -adrenoceptor antagonist) and ketanserin but not significantly inhibited by spiperone and methysergide. KCl has a weak potency for producing a vasoconstriction of the canine common carotid artery. A relatively large dose of diltiazem (a calcium channel blocker) did not modify 5-HT-induced vasoconstrictions. From these results, we conclude that (a) the canine common carotid artery contains abundant 5-HT receptors; (b) there are no functional 5-HT₁ receptors, but 5-HT₂ receptors are prominent; (c) 5-CT-induced vasoconstrictions might be due to activation of 5-HT₂ but not to 5-HT₁ receptors; (d) 5-HT-induced vasoconstriction might not involve -adrenoceptors; and (e) the vasoconstriction related to 5-HT in the common carotid artery is not significantly mediated via activation of calcium ion channels of smooth muscle cells, but may be induced by calcium ions from intracellular stores.     

β2受体对心肌缝隙连接蛋白Cx43的调节

目的探讨β-肾上腺素受体（β-AR）亚型对心肌缝隙连接蛋白connexin43（Cx43）表达及功能的调节。方法采用免疫印迹技术和划痕标记示踪技术（SLDT）观察心肌细胞Cx43的表达和功能变化。结果给予异丙基肾上腺素（ISO）刺激5min，即可明显增加心肌细胞非磷酸化Cx43的表达，同时促进荧光染料在细胞间的扩散；选择性β2-AR拮抗剂ICI 118551，可完全拮抗异丙基肾上腺素所诱导的该作用。结论异丙基肾上腺素主要通过刺激BrAR参与调控Cx43，这可能是β2-AR诱导心律失常的一个重要机制。     

Genetic deletion of MAO-A promotes serotonin-dependent ventricular hypertrophy by pressure overload

The potential role of serotonin (5-HT) in cardiac function has generated much interest in recent years. In particular, the need for a tight regulation of 5-HT to maintain normal cardiovascular activity has been demonstrated in different experimental models. However, it remains unclear how increased levels of 5-HT could contribute to the development of cardiac hypertrophy. Availability of 5-HT depends on the mitochondrial enzyme monoamine oxidase A (MAO-A). Therefore, we investigated the consequences of MAO-A deletion on ventricular remodeling in the model of aortic banding in mice. At baseline, MAO-A deletion was associated with an increase in whole blood 5-HT (39.4 ± 1.9 μM vs. 24.0 ± 0.9 μM in KO and WT mice, respectively). Cardiac 5-HT_2A, but not 5-HT_2B receptors were overexpressed in MAO-A KO mice, as demonstrated by real-time PCR and Western-blot experiments. After aortic banding, MAO-A KO mice demonstrated greater increase in heart wall thickness, heart to body weight ratios, cardiomyocyte cross-section areas, and myocardial fibrosis compared to WT. Exacerbation of hypertrophy in KO mice was associated with increased amounts of 5-HT in the heart. In order to determine the role of 5-HT and 5-HT_2A receptors in ventricular remodeling in MAO-A KO mice, we administered the 5-HT_2A receptor antagonists ketanserin (1 mg/kg/day) or M100907 (0.1 mg/kg/day) during 4 weeks of aortic banding. Chronic administration of these antagonists strongly prevented exacerbation of ventricular hypertrophy in MAO-A KO mice. These results show for the first time that regulation of peripheral 5-HT by MAO-A plays a role in ventricular remodeling via activation of 5-HT_2A receptors.     

Serotonin receptor subtypes influence prolactin secretion in the turkey

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D₁ dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(−)-DOI hydrochloride (DOI), a selective 5-HT_2A eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT_2A receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D₁ DA receptors in the INF were blocked by the D₁ DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT_2A receptor subtype. The DOI-induced PRL increase did not occur when (±)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T_1A receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT_2A receptors are activated, they influence the release of DA to the INF. When 5-HT_1A receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT_2A receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D₂ DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT_1A receptors, could effectively inhibit PRL secretion.     

Serodolin,a β-arrestin–biased ligand of 5-HT7 receptor,attenuates pain-related behaviors

G protein–coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT₇ receptor (5-HT₇R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on G_s signaling while inducing ERK activation through a β-arrestin–dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT₇R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT₇R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT₇R stimulation have been classically shown to result from G_s-dependent adenylyl cyclase activation. In this study, using a β-arrestin–biased agonist, we provided insight into the molecular mechanism triggered by 5-HT₇R and revealed its therapeutic potential in the modulation of pain response.

Among 14 serotonin receptor subtypes, 5-hydroxytryptamine 7receptors (5-HT₇Rs) belong to the G protein–coupled receptor (GPCR) family or the so-called seven transmembrane-spanning receptor. It is the last identified member and has been cloned from several animal species, including human (1). 5-HT₇R couples to the heterotrimeric G protein G_s, which in turn stimulates adenylate cyclase (AC), leading to an increase of 3′-5′-cyclic adenosine monophosphate (cAMP) production in both recombinant and native systems (1). This allows the activation of cAMP-dependent protein kinase (PKA), which acts on the MAPK cascade in a cell type–specific manner (2–4).In HEK-293 cells, the observed agonist-induced ERK1/2 activation requires PKA, Ras, and Raf activation independently of Rap-1 (4). In neurons, the 5-HT₇R–induced ERK activation is mediated through a PKA-independent pathway that utilizes cAMP-guanine nucleotide exchange factor (cAMP-GEF) (3). It was shown that 5-HT₇R signaling also depends on the activation of Gα₁₂ protein that in turn triggers activation of multiple signaling pathways through the family of small Rho GTPases, Cdc42 and RhoA (5).The 5-HT₇R is expressed in the peripheral and central nervous system with highest densities in thalamus, hypothalamus, cerebral cortex, amygdala, and striatal complex (1). Numerous data have established 5-HT₇R implication in the control of circadian rhythms and thermoregulation, learning, and memory as well as in central nervous system (CNS) disorders such as depression, Alzheimer’s disease, and schizophrenia (6). There is mounting evidence that 5-HT₇R is an important modulator of nociceptive transmission (7). It was also reported that 5-HT₇R is involved in the antinociceptive effects of morphine (8), antidepressants, and nonopioid analgesics (9). Collectively, these observations underscore the interest of developing new 5-HT₇R ligands for the treatment of pain. To date, many 5-HT₇R potent agonists (5-CT, E55888, AS-19, and LP-211) and antagonists (SB269-970 and DR4004) against 5-HT₇R have been described (10); these are all ligands that commit the receptor to a G protein–dependent pathway, although few 5-HT₇R–biased ligands have been described (11, 12).In contrast to standard agonists and antagonists, which activate or inactivate the entirety of a receptor’s signaling network, some ligands termed biased ligands are capable of stabilizing subsets of receptor conformations, hence eliciting selective modulation within the network. From data obtained in the last two decades, the concept of functional selectivity of a ligand has emerged as an interesting property in modern drug discovery. Increasing preclinical data highlight the value of using such ligands, which exhibit a unique spectrum of pharmacological responses, for instance, by specifically targeting G protein– or β-arrestin–dependent signaling. Biased ligands, by selectively modulating a subset of receptor functions, may optimize therapeutic action and generate less pronounced side effects than compounds globally affecting receptor activity. Although binding of β-arrestins to GPCR has primarily been involved in the termination of G protein signaling by inducing desensitization and internalization of the receptor, numerous studies have indicated that β-arrestins can be intimately involved in additional signaling events through mechanisms dependent on or independent of G protein coupling (13, 14). Several β-arrestin–biased ligands have been identified and show therapeutic interest in other receptor classes (15). In particular, several studies demonstrate the role of β-arrestin signaling on opioid analgesia and tolerance (16, 17). In the present study, we used a combination of pharmacological, genetic, and behavioral approaches to identify a β-arrestin–biased 5-HT₇R ligand and evaluate its therapeutical potential for the treatment of pain.     

Chronic 5-HT2-Receptor Blockade by Ritanserin Does Not Reduce Blood Pressure in Patients with Essential Hypertension

ABSTRACT The selective 5-HT₂-receptor-blocking agent ritanserin is an analogue of the antihypertensive agent ketanserin. By evaluating the antihypertensive effects of ritanserin the aim of this investigation was to indirectly elucidate the mechanism of action of ketanserin. Thirteen patients with essential hypertension were treated with placebo and ritanserin, 10 mg bid., in a double-blind, cross-over design (4-week periods). At the end of the treatment periods blood pressure as well as plasma concentrations of ritanserin were evaluated for 24 hours. Despite high steady state and peak plasma concentrations of ritanserin the compound did not lower the blood pressure compared with placebo. Since chronic selective 5-HT₂-receptor blockade by means of ritanserin did not lower the blood pressure, it is concluded that the 5-HT₂ blocking properties of ketanserin cannot alone be responsible for the antihypertensive effects of ketanserin.     

Perturbed dentate gyrus function in serotonin 5-HT2C receptor mutant mice

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT_2C receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT_2C receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT_2C-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path–dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT_2C receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.     

Gq/5-HT2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice

Serotonin 2c receptors (5-HT_2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT_2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT_2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT_2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT_2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT_2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT_2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.Serotonin (5-hydroxytryptamine, or 5-HT) is an important modulator of anxiety circuits (1). The diverse effects of serotonin are mediated through various 5-HT receptors (5-HT-Rs), including 5-HT_1–7-Rs (2). Recent pharmacologic and genetic studies have highlighted an important role of 5-HT_2c-Rs in anxiety disorders; however, the interpretation of physiological and behavioral data remains difficult owing to a lack of selective pharmacologic ligands (3).5-HT_2c-Rs are expressed in various cell types and brain regions of the anxiety circuit, including the amygdala and the dorsal raphe nucleus (DRN), a midbrain region containing high concentrations of 5-HT neurons. It has been suggested that 5-HT_2c-Rs are expressed in GABAergic neurons, and that 5-HT_2c-R activation may contribute to an inhibitory feedback control of 5-HT cell firing (4). The functional and behavioral consequences of such a possible inhibitory feedback mechanism for 5-HT firing have not yet been investigated, however.Unfortunately, current techniques for identifying the functions of 5-HT_2c-Rs in vertebrate brains are of limited value. For example, agonists and antagonists of 5-HT_2c-Rs are often unspecific, and their action is not restricted to a specific cell type. Complete and conditional knockouts of the receptor gene have limited control of developmental and compensation effects by other G-protein–coupled receptors (GPCRs), and none of the current techniques allows for the physiological control of the 5-HT_2c-R activation on a millisecond to second time scale.To overcome the limitations of pharmacologic and genetic approaches, we have developed a new light-activated GPCR based on vertebrate melanopsin (vMo). Both 5-HT_2c-Rs and vMo couple to the Gq signaling pathway (5, 6). To investigate the functional consequence of Gq signal activation in the cell types and cellular structures where 5-HT_2c-Rs are located, we virally expressed vMo carrying the C terminus (CT) of the 5-HT_2c-R in GABAergic neurons in the DRN. We found that light activation of vMo-CT_5-HT2c decreases the firing of 5-HT neurons and modulates anxiety behaviors in mice. Our results demonstrate a previously unidentified, autoregulatory negative feedback mechanism for the firing of serotonergic neurons to control anxiety in mice.     

Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling

The 5-HT_2A serotonin receptor is the most abundant serotonin receptor subtype in the cortex and is predominantly expressed in pyramidal neurons. The 5-HT_2A receptor is a target of several hallucinogens, antipsychotics, anxiolytics, and antidepressants, and it has been associated with several psychiatric disorders, conditions that are also associated with aberrations in dendritic spine morphogenesis. However, the role of 5-HT_2A receptors in regulating dendritic spine morphogenesis in cortical neurons is unknown. Here we show that the 5-HT_2A receptor is present in a subset of spines, in addition to dendritic shafts. It colocalizes with PSD-95 and with multiple PDZ protein-1 (MUPP1) in a subset of dendritic spines of rat cortical pyramidal neurons. MUPP1 is enriched in postsynaptic density (PSD) fractions, is targeted to spines in pyramidal neurons, and enhances the localization of 5-HT_2A receptors to the cell periphery. 5-HT_2A receptor activation by the 5-HT₂ receptor agonist DOI induced a transient increase in dendritic spine size, as well as phosphorylation of p21-activated kinase (PAK) in cultured cortical neurons. PAK is a downstream target of the neuronal Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 that is important for spine remodeling. Kalirin-7 regulates dendritic spine morphogenesis in neurons but its role in neuromodulator signaling has not been investigated. We show that peptide interference that prevents the localization of kalirin-7 to the postsynaptic density disrupts DOI-induced PAK phosphorylation and spine morphogenesis. These results suggest a potential role for serotonin signaling in modulating spine morphology and kalirin-7''s function at cortical synapses.     

Promiscuous coassembly of serotonin 5-HT3 and nicotinic α4 receptor subunits into Ca2+-permeable ion channels

Serotonin (5-hydroxytryptamine) type 3 receptors (5-HT₃R) and nicotinic acetylcholine receptors are structurally and functionally related proteins, yet distinct members of the family of ligand-gated ion channels. For most members of this family a diversity of heteromeric receptors is known at present. In contrast, known 5-HT₃R subunits are all homologs of the same 5-HT₃R-A subunit and form homopentameric receptors. Here we show, by heterologous expression followed by immunoprecipitation, that 5-HT₃R and nicotinic acetylcholine receptor α4 subunits coassemble into a novel type of heteromeric ligand-gated ion channel, which is activated by 5-HT. The Ca²⁺ permeability of this heteromeric ion channel is enhanced as compared with that of the homomeric 5-HT₃R channel. Heteromeric 5-HT₃/α4 and homomeric 5-HT₃Rs have similar pharmacological profiles, but distinct sensitivities to block by the antagonist d-tubocurarine. Coassembly of subunits beyond the boundaries of ligand-gated ion channel families may constitute an important mechanism contributing to the diverse properties and functions of native neurotransmitter receptors.     

Serotonin Agonists and Antagonists in Obstructive Sleep Apnea

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a prevalent disorder associated with substantial cardiovascular and neurobehavioral morbidity. Yet this is a disorder for which there are no widely effective pharmacotherapies. The pathophysiology of obstructive sleep apnea namely, normal respiration in waking with disordered breathing only in sleep, suggests that this disorder should be readily amenable to drug therapy. Over the past 10 years, we have gained tremendous insight into the neurochemical mechanisms involved in state-dependent control of respiration. It is apparent from this work that there are many potential avenues for pharmacotherapies, including several seemingly conflicting directions for serotonergic therapies.Serotonin delivery is reduced to upper airway dilator motor neurons in sleep, and this contributes, at least in part, to sleep-related reductions in dilator muscle activity and upper airway obstruction. The dilator motor neuron post-synaptic serotonin receptors are 5-HT_2A and 5-HT_2C subtypes, and in adults the presynaptic 5-HT receptor in motor nuclei is 5-HT_1B, an inhibitory receptor. Serotonin receptors are also found within central respiratory neuronal groups, and these receptor subtypes include 5-HT_1A (inhibitory) and 5-HT₂ receptors. Peripherally, stimulation of 5-HT_2A, 5-HT_2C and 5-HT₃ receptor subtypes have an inhibitory effect on respiration via action at the nodose ganglion. Many of these receptor subtypes and their signal transduction pathways may be affected by oxidative stress in obstructive sleep apnea. These alterations will make finding drug therapies for sleep apnea more challenging, but not insurmountable. Future directions are suggested for elucidating safe, well-tolerated serotonergic drugs for this disorder.Tryptophan was one of the first serotonergic drugs tested for OSAHS. This drug was withdrawn from the market as a result of reports linking tryptophan use with eosinophilic myalgia syndrome and life-threatening pulmonary hypertension. Newer drugs with serotonergic activity tested in persons with sleep-disordered breathing include buspirone, fluoxetine and paroxetine. Trials are presently being conducted to evaluate the effects of 5-HT_2A and 5-HT₃ antagonists on OSAHS. Many of the drugs tested have not shown significant improvement in sleep apnea. However, with continued effort to elucidate the pharmacology of neurochemical control of state-dependent changes in respiratory control, the availability of pharmacological therapy for this disorder is not too far away.     

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [³H]-5-HT-moduline bound specifically to rat brain sections with high affinity (K_d = 0.77 nM and B_max = 0.26 dpm/mm²). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT_1B receptor knock-out mice, the specific binding of [³H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT_1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT_1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT_1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT_1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT_1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT_1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.     

Increased Expression of 5-HT3 and NK1 Receptors in 5-Fluorouracil-Induced Mucositis in Mouse Jejunum

Background and Objective

Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80 % of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT₃ and NK₁ receptor expression by immunohistochemical analysis.

Methods

5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay.

Results

The numbers of 5-HT₃ receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT₃ receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK₁ receptor cells in mucosa and immunopositive expression of NK₁ receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT_3A, 5-HT_3B, and NK₁ receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT₃ or NK₁ receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT₃ and NK₁ receptors colocalized with CD11b.

Conclusions

The 5-HT₃ and NK₁ immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.     

Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT_1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT_1A receptor-mutant mice. These animals lack functional 5-HT_1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT_1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT_1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.     

5-HT 2A Receptor and 5-HT Degradation Play a Crucial Role in Atherosclerosis by Modulating Macrophage Foam Cell Formation,Vascular Endothelial Cell Inflammation,and Hepatic Steatosis

Aim: Previously, we found that diabetes-related liver dysfunction is due to activation of the 5-HT _2A receptor (5-HT _2A R) and increased synthesis and degradation of 5-HT. Here, we investigated the role of 5-HT in the development of atherosclerosis. Methods: The study was conducted using high-fat diet-fed male ApoE ^−/− mice, THP-1 cell-derived macrophages, and HUVECs. Protein expression and biochemical indexes were determined by Western blotting and quantitative analysis kit, respectively. The following staining methods were used: oil red O staining (showing atherosclerotic plaques and intracellular lipid droplets), immunohistochemistry (showing the expression of 5-HT _2A R, 5-HT synthase, and CD68 in the aortic wall), and fluorescent probe staining (showing intracellular ROS). Results: In addition to improving hepatic steatosis, insulin resistance, and dyslipidemia, co-treatment with a 5-HT synthesis inhibitor and a 5-HT _2A R antagonist significantly suppressed the formation of atherosclerotic plaques and macrophage infiltration in the aorta of ApoE ^−/− mice in a synergistic manner. Macrophages and HUVECs exposed to oxLDL or palmitic acid in vitro showed that activated 5-HT _2A R regulated TG synthesis and oxLDL uptake by activating PKCε, resulting in formation of lipid droplets and even foam cells; ROS production was due to the increase of both intracellular 5-HT synthesis and mitochondrial MAO-A-catalyzed 5-HT degradation, which leads to the activation of NF-κB and the release of the inflammatory cytokines TNF-α and IL-1β from macrophages and HUVECs as well as MCP-1 release from HUVECs. Conclusion: Similar to hepatic steatosis, the pathogenesis of lipid-induced atherosclerosis is associated with activation of intracellular 5-HT _2A R, 5-HT synthesis, and 5-HT degradation.     

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HT_apAC1, stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HT_apAC1 expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HT_apAC1 as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.