Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

Modulation of Prostaglandin G/H Synthase Expression in Mesangial Cells Transfected by pp60Proto-oncogene

The role of the protein tyrosine kinase pp60^c-srcin the expression of prostaglandin G/H synthase (PGHS), the key enzyme of prostaglandin synthesis, was investigated in rat renal mesangial cells. Transfection of mesangial cells with the proto-oncogene c-src resulted in nontransformed cells with constitutively enhanced pp60^c-srckinase activity. As a control, mesangial cells were transfected with inactive pp60^c-src, mutated in position 295 (lysine replaced by methionine). Expression of the constitutive isoform PGHS-1 was enhanced in cells overexpressing wild-type c-src compared to cells transfected with the kinase negative c-src mutant. Levels of other constitutively expressed proteins such as GAPDH and β-actin were also enhanced. PGHS-2 was barely detectable in resting cells but was inducible by PDGF-AB, PDGF-BB, serotonin, FCS, and calcium ionophore A23187. Induction was diminished in pp60^c-srckinase-overexpressing cells, independent of the stimulus used, suggesting interference at a late step in the signaling cascade. No induction of PGHS-2 mRNA was observed in mesangial cells transformed by the oncogene v-src. An increase in intracellular calcium levels is an early step in signal transduction by PDGF and serotonin in mesangial cells. c-src kinase overexpression reduced PDGF- and serotonin-mediated changes in Ca²⁺signaling, indicating multiple targets of pp60^c-srcaction. Overexpression of pp60^c-srcin mesangial cells thus affected basal protein expression, reflected by the enhanced PGHS-1 mRNA and protein expression. With regard to induction of PGHS-2, overexpression of pp60^c-srcreduced induction by stimuli coupled to different types of signaling pathways.     

Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

Stimulation of 5-HT₃ receptors (5-HT₃Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT₃ receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT₃R channel has high permeability to extracellular calcium (Ca²⁺) and upon stimulation allows increased Ca²⁺ influx. We examined the contribution of Ca²⁺/calmodulin-dependent protein kinase IIα (Ca²⁺/CaMKIIα), interaction of 5-HT₃R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca²⁺ levels in brainstem slices and that the selective 5-HT₃R antagonist palonosetron, can abolish the induced Ca²⁺ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca²⁺ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca²⁺-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT₃R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca²⁺ mobilization via extracellular Ca²⁺ influx through 5-HT₃Rs/L-type Ca²⁺ channels, and intracellular Ca²⁺ release via RyRs on ER, initiate Ca²⁺-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT₃R-mediated, 2-Me-5-HT-evoked emesis.     

Serotonin Regulation of Serotonin Uptake in RN46A Cells

1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT_1A, 5-HT_1B, 5-HT_2A, and 5-HT_2C) and as cAMP and intracellular Ca²⁺ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [³H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca²⁺ changes were determined using the ratiometric method of intracellular Ca²⁺ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [³H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca²⁺ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca²⁺ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca²⁺ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca²⁺ levels. This suggests that 5-HT may utilize intracellular Ca²⁺ to regulate 5-HT uptake.     

Effects of sarpogrelate on serotonin-induced increases in cytosolic Ca2+ in cultured rat mesangial cells

The effects of the new 5-HT_2A receptor antagonist sarpogrelate on the cellular action of serotonin were examined in cultured rat mesangial cells by measuring cytosolic free calcium concentration ([Ca²⁺]i). Sarpogrelate inhibited serotonin-induced increases in [Ca²⁺]i in a concentration-dependent manner. M1, a major metabolite of sarpogrelate, also exhibited an inhibitory effect exceeding that of sarpogrelate. The inhibitory effects of sarpogrelate and M1 were abolished by washing out these compounds. In contrast, the increase in [Ca²⁺]i induced by angiotensin II or arginine vasopressin was not affected by pretreatment of the cells with sarpogrelate or M1. These results suggest that sarpogrelate and its major metabolite (M1) act as reversible and specific 5-HT_2A receptor antagonists against the contractile action of platelet-derived serotonin in mesangial cells.     

PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT<Subscript>1A</Subscript> Receptor and Upregulation of Nrf2-Dependent GSH Synthesis

We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT_1A receptor, but not 5-HT_2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT_1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT_1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT_1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT_1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT_1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT_1A receptor and upregulation of Nrf2-dependent GSH synthesis.     

Substitution of 5-HT1A Receptor Signaling by a Light-activated G Protein-coupled Receptor

Understanding serotonergic (5-HT) signaling is critical for understanding human physiology, behavior, and neuropsychiatric disease. 5-HT mediates its actions via ionotropic and metabotropic 5-HT receptors. The 5-HT_1A receptor is a metabotropic G protein-coupled receptor linked to the G_i/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT_1A signaling, we created a light-activated G protein-coupled receptor that targets into 5-HT_1A receptor domains and substitutes for endogenous 5-HT_1A receptors. To induce 5-HT_1A-like targeting, vertebrate rhodopsin was tagged with the C-terminal domain (CT) of 5-HT_1A (Rh-CT_5-HT1A). Rh-CT_5-HT1A activates G protein-coupled inward rectifying K⁺ channels in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT_1A receptor. The intracellular distribution of Rh-CT_5-HT1A resembles that of the 5-HT_1A receptor; Rh-CT_5-HT1A localizes to somatodendritic sites and is efficiently trafficked to distal dendritic processes. Additionally, neuronal expression of Rh-CT_5-HT1A, but not Rh, decreases 5-HT_1A agonist sensitivity, suggesting that Rh-CT_5-HT1A and 5-HT_1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT_5-HT1A is able to rescue 5-HT_1A signaling of 5-HT_1A KO mice in cultured neurons and in slices of the dorsal raphe showing that Rh-CT_5-HT1A is able to functionally compensate for native 5-HT_1A. Thus, as an optogenetic tool, Rh-CT_5-HT1A has the potential to directly correlate in vivo 5-HT_1A signaling with 5-HT neuron activity and behavior in both normal animals and animal models of neuropsychiatric disease.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

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.     

Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling

The serotonin (5-hydroxytryptamine; 5-HT)_2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT_2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca²⁺-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT_2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT_2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT_2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT_2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT_2C receptor agonists and their antidepressant-like activity.     

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.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

5-hydroxytryptamine has an endothelium-derived hyperpolarizing factor-like effect on coronary flow in isolated rat hearts

Background

5-hydroxytryptamine (5-HT)-induced coronary artery responses have both vasoconstriction and vasorelaxation components. The vasoconstrictive effects of 5-HT have been well studied while the mechanism(s) of how 5-HT causes relaxation of coronary arteries has been less investigated. In isolated rat hearts, 5-HT-induced coronary flow increases are partially resistant to the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) and are blocked by 5-HT₇ receptor antagonists. In the present study, we investigated the role of 5-HT₇ receptor in 5-HT-induced coronary flow increases in isolated rat hearts in the absence of L-NAME, and we also evaluated the involvement of endothelium-derived hyperpolarizing factor (EDHF) in 5-HT-induced coronary flow increases in L-NAME-treated hearts with the inhibitors of arachidonic acid metabolism and the blockers of Ca²⁺-activated K⁺ channels.

Results

In isolated rat hearts, 5-HT and the 5-HT₇ receptor agonist 5-carboxamidotryptamine induced coronary flow increases, and both of these effects were blocked by the selective 5-HT₇ receptor antagonist SB269970; in SB269970-treated hearts, 5-HT induced coronary flow decreases, which effect was blocked by the 5-HT_2A receptor blocker {"type":"entrez-nucleotide","attrs":{"text":"R96544","term_id":"982204","term_text":"R96544"}}R96544. In L-NAME-treated hearts, 5-HT-induced coronary flow increases were blocked by the phospholipase A₂ inhibitor quinacrine and the cytochrome P450 inhibitor SKF525A, but were not inhibited by the cyclooxygenase inhibitor indomethacin. As to the effects of the Ca²⁺-activated K⁺ channel blockers, 5-HT-induced coronary flow increases in L-NAME-treated hearts were inhibited by TRAM-34 (intermediate-conductance Ca²⁺-activated K⁺ channel blocker) and UCL1684 (small-conductance Ca²⁺-activated K⁺ channel blocker), but effects of the large-conductance Ca²⁺-activated K⁺ channel blockers on 5-HT-induced coronary flow increases were various: penitrem A and paxilline did not significantly affect 5-HT-induced coronary flow responses while tetraethylammonium suppressed the coronary flow increases elicited by 5-HT.

Conclusion

In the present study, we found that 5-HT-induced coronary flow increases are mediated by the activation of 5-HT₇ receptor in rat hearts in the absence of L-NAME. Metabolites of cytochrome P450s, small-conductance Ca²⁺-activated K⁺ channel, and intermediate-conductance Ca²⁺-activated K⁺ channel are involved in 5-HT-induced coronary flow increases in L-NAME-treated hearts, which resemble the mechanisms of EDHF-induced vasorelaxation. The role of large-conductance Ca²⁺-activated K⁺ channel in 5-HT-induced coronary flow increases in L-NAME-treated hearts needs further investigation.     

p11 modulates calcium handling through 5-HT4R pathway in rat ventricular cardiomyocytes

BackgroundThe role of the serotonin receptor 4 (5-HT₄R) pathway in cardiac excitation-contraction coupling (ECC) remains unclear. In the brain, induction of the calcium (Ca²⁺)-binding protein p11 enhances 5-HT₄R translocation and signaling and could therefore be considered as a modulator of the 5-HT₄R pathway in the myocardium. p11 expression is increased by brain-derived neurotrophic factor (BDNF) or antidepressant drugs (imipramine). Thus, we investigated whether p11 regulates the 5-HT₄R pathway in the heart in physiological conditions or under pharmacological induction and the effects on calcium handling.Methods and resultsp11 expression was induced in vivo in healthy Wistar rats by imipramine (10 mg/kg/21 days) and in vitro in left ventricular cardiomyocytes exposed to BDNF (50 ng/ml/8 h). Cell shortening and real-time Ca²⁺ measurements were processed on field-stimulated intact cardiomyocytes with the selective 5-HT₄R agonist, prucalopride (1 μM). Both imipramine and BDNF-induced cardiomyocyte p11 expression unmasked a strong response to prucalopride characterized by an increase of both cell shortening and Ca²⁺ transient amplitude compared to basal prucalopride associated with a high propensity to trigger diastolic Ca²⁺ events. Healthy rats treated with BDNF (180 ng/day/14 days) exhibited a sustained elevated heart rate following a single injection of prucalopride (0.1 mg/kg) which was not observed prior to treatment.ConclusionsWe have identified a novel role for p11 in 5-HT₄R signaling in healthy rat ventricular cardiomyocytes. Increased p11 expression by BDNF and imipramine unraveled a 5-HT₄R-mediated modulation of cardiac Ca²⁺ handling and ECC associated with deleterious Ca²⁺ flux disturbances. Such mechanism could partly explain some cardiac adverse effects induced by antidepressant treatments.     

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.     

Inactivation of 5-HT1A and [3H]5-HT Binding Sites by N-Ethoxycarbonyl-2-Ethoxy-1, 2-Dihydroquinoline (EEDQ) in Rat Brain

Inactivation of 5-HT_1A and [³H]5-HT binding sites by N-Ethoxycarbonyl-2-ethoxy-1, 2-dihydro-quinoline (EEDQ) was studied in regions of rat brain. After exposure to EEDQ (4 mg/kg body wt.) for 7 days, it is observed that the density of 5-HT₁ receptor sites was decreased by nearly 20% in both cortex and hippocampus. The decrease, however, in 5-HT_1A sites was more significant (70%) in both the regions. The affinity of [³H]5-HT to 5-HT₁ sites was decreased significantly in both cortex and hippocampus after exposure to EEDQ, without affecting the Kd of 5-HT_1A sites. Displacement studies suggested that EEDQ has high affinity to 5-HT₁ sites with a Ki of 42.9 ± 2.4 nM. After exposure neither basal nor 5-HT stimulated adenylyl cyclase activity was changed in cortex. The results of this study suggest that EEDQ decreases the density of 5-HT₁ and 5-HT_1A receptor sites but does not cause functional downregulation of these sites in rat brain.