Cloning and Expression of a Human Serotonin 5-HT4 Receptor cDNA

Abstract: Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT₄ receptor sequence, we have cloned the complete coding region for a human 5-HT₄ receptor. The sequence shows extensive similarity to the published porcine 5-HT_4A and rat 5-HT_4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT₄ sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT₄ receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT₄ receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT₄ receptors in guinea pig striatal tissue. [³H]GR 113808 revealed K _D values of 0.15 ± 0.01 n M for the human receptor and 0.3 ± 0.1 n M in the guinea pig tissue. Binding constants were determined for four investigated 5-HT₄ antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT₄-specific agonists caused an increase in cyclic AMP levels.     

The Third Intracellular Loop of the 5-Hydroxytryptamine2A Receptor Determines Effector Coupling Specificity

Abstract: 5-Hydroxytryptamine (5-HT) receptors contain seven putative transmembrane domains and couple via different guanine nucleotide binding proteins to specific effector enzymes. Studies with other receptors identify the second and third intracellular loops or the C-terminus of the receptor as important for selective effector coupling. However, it is not known which regions of the 5-HT receptor determine effector coupling specificity. To address this question, we constructed a chimeric 5-HT receptor in which the third intracellular (i3) loop is derived from the 5-HT_2A receptor, which is coupled to activation of phospholipase C, and the rest of the sequence is derived from the 5-HT_1B receptor, which is coupled to inhibition of adenylyl cyclase. The chimeric receptor exhibited ligand binding properties similar to those of the 5-HT_1B receptor and distinct from those of the 5-HT_2A receptor. This suggests that the i3 loop is not critical for the unique pharmacology of the 5-HT_1B receptor. In contrast, the chimeric receptor exhibited signaling properties similar to those of the 5-HT_2A receptor and distinct from those of the 5-HT_1B receptor. This indicates that the i3 loop determines the effector coupling specificity of the 5-HT_2A receptor.     

Differential Regulation of Somatodendritic Serotonin 5-HT1A Receptors by 2-Week Treatments with the Selective Agonists Alnespirone (S-20499) and 8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract : Single treatment with the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) and alnespirone (S-20499) reduces the extracellular 5-HT concentration (5-HT_ext) in the rat midbrain and forebrain. Given the therapeutic potential of selective 5-HT_1A agonists in the treatment of affective disorders, we have examined the changes in 5-HT_1A receptors induced by 2-week minipump administration of alnespirone (0.3 and 3 mg/kg/day) and 8-OH-DPAT (0.1 and 0.3 mg/kg/day). The treatment with alnespirone did not modify baseline 5-HT_ext but significantly attenuated the ability of 0.3 mg/kg s.c. alnespirone to reduce 5-HT_ext in the dorsal raphe nucleus (DRN) and frontal cortex. In contrast, the ability of 8-OH-DPAT (0.025 and 0.1 mg/kg s.c.) to reduce 5-HT_ext in both areas was unchanged by 8-OH-DPAT pretreatment. Autoradiographic analysis revealed a significant reduction of [³H]8-OH-DPAT and [³H]WAY-100635 {³H-labeled N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexanecarboxamide · 3HCl} binding to somatodendritic 5-HT_1A receptors (but not to postsynaptic 5-HT_1A receptors) of rats pretreated with alnespirone but not with 8-OH-DPAT. In situ hybridization analysis revealed no change of the density of the mRNA encoding the 5-HT_1A receptors in the DRN after either treatment. These data indicate that continuous treatment for 2 weeks with alnespirone, but not with 8-OH-DPAT, causes a functional desensitization of somatodendritic 5-HT_1A receptors controlling 5-HT release in the DRN and frontal cortex.     

Differential Membrane Targeting and Pharmacological Characterization of Chimeras of Rat Serotonin 5-HT1A and 5-HT1B Receptors Expressed in Epithelial LLC-PK1 Cells

Abstract: The serotonin 5-HT_1A and 5-HT_1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT_1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT_1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT_1A and 5-HT_1B receptors still able to bind specifically [³H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT_1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT_1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT_1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT_1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).     

Preferential Potentiation of the Effects of Serotonin Uptake Inhibitors by 5-HT1A Receptor Antagonists in the Dorsal Raphe Pathway: Role of Somatodendritic Autoreceptors

Abstract: 5-HT_1A autoreceptor antagonists enhance the effects of antidepressants by preventing a negative feedback of serotonin (5-HT) at somatodendritic level. The maximal elevations of extracellular concentration of 5-HT (5-HT_ext) induced by the 5-HT uptake inhibitor paroxetine in forebrain were potentiated by the 5-HT_1A antagonist WAY-100635 (1 mg/kg s.c.) in a regionally dependent manner (striatum > frontal cortex > dorsal hippocampus). Paroxetine (3 mg/kg s.c.) decreased forebrain 5-HT_ext during local blockade of uptake. This reduction was greater in striatum and frontal cortex than in dorsal hippocampus and was counteracted by the local and systemic administration of WAY-100635. The perfusion of 50 µmol/L citalopram in the dorsal or median raphe nucleus reduced 5-HT_ext in frontal cortex or dorsal hippocampus to 40 and 65% of baseline, respectively. The reduction of cortical 5-HT_ext induced by perfusion of citalopram in midbrain raphe was fully reversed by WAY-100635 (1 mg/kg s.c.). Together, these data suggest that dorsal raphe neurons projecting to striatum and frontal cortex are more sensitive to self-inhibition mediated by 5-HT_1A autoreceptors than median raphe neurons projecting to the hippocampus. Therefore, potentiation by 5-HT_1A antagonists occurs preferentially in forebrain areas innervated by serotonergic neurons of the dorsal raphe nucleus.     

Regulation of Serotonin2A Receptor Expression by an Antisense Oligodeoxynucleotide

Abstract: The regulation of 5-HT_2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT_2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A₁A₁ variant cells, which express the 5-HT_2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT_2A receptor sites as measured by the binding of 2-[¹²⁵I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT_2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT_2A receptor density and an increase in headshake behavior induced by the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT_2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT_2A receptor expression.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Mianserin-Induced Down-Regulation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH-SY5Y

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5-hydroxytryptamine_2A (5-HT_2A) and 5-HT_2C receptors stably expressed in the human neuroblastoma cell line SH-SY5Y. Incubation of cells expressing the 5-HT_2A receptor with mianserin (100 n M ) for 24 h caused a significant decrease (48%) in the binding capacity of [³H]ketanserin. This receptor down-regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5-HT but not by carbachol. Exposure of cells expressing the 5-HT_2C receptor to mianserin (100 n M ) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [³H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5-HT at the 5-HT_2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down-regulation is evident at human 5-HT_2A and 5-HT_2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.     

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5-HT₆ serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT₆ cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT₆ receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT₆ amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT₆ receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5-HT_1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT₆ receptor also reveals an Rsa I restriction fragment length polymorphism within the coding region.     

The brain 5-HT4 receptor binding is down-regulated in the Flinders Sensitive Line depression model and in response to paroxetine administration

The 5-hydroxytryptamine (5-HT₄) receptor may be implicated in depression and is a new potential target for antidepressant treatment. We have investigated the brain 5-HT₄ receptor [³H]SB207145 binding in the Flinders Sensitive Line rat depression model by quantitative receptor autoradiography, and related this to 5-HT transporter ( S )-[ N -methyl-³H]citalopram binding. We also determined the regulation of 5-HT₄ receptor binding by 1, 14, and 21 days of paroxetine administration and subchronic 5-HT depletion, and compared this with changes in 5-HT_2A receptor [³H]MDL100907 binding. In the Flinders Sensitive Line, the 5-HT₄ receptor and 5-HT transporter binding were decreased in the dorsal and ventral hippocampus, and the changes in binding were directly correlated within the dorsal hippocampus. Chronic but not acute paroxetine administration caused a 16–47% down-regulation of 5-HT₄ receptor binding in all regions evaluated including the basal ganglia and hippocampus, while 5-HT depletion increased the 5-HT₄ receptor binding in the dorsal hippocampus, hypothalamus, and lateral globus pallidus. In comparison, the 5-HT_2A receptor binding was decreased in the frontal and cingulate cortices after chronic paroxetine administration, and markedly reduced in several regions after 5-HT depletion. Thus, the 5-HT₄ receptor binding was decreased in the Flinders Sensitive Line depression model and in response to chronic paroxetine administration.     

Activating Mutations of the Serotonin 5-HT2C Receptor

Abstract: Site-directed mutagenesis was performed to create a mutant serotonin 5-HT_2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [³H]-mesulergine-labeled 5-HT_2C receptors increased from 203 n M (native) to 76 n M for S312F and 6.6 n M for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 n M (native) to 28 n M for S312F and 2.7 n M for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [³H]5-HT and [³H]-mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [³H]5-HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT_2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.     

Potentiation of the Fluoxetine-Induced Increase in Dialysate Levels of Serotonin (5-HT) in the Frontal Cortex of Freely Moving Rats by Combined Blockade of 5-HT1A and 5-HT1B Receptors with WAY 100,635 and GR 127,935

Abstract: In this study, we examined the influence of blockade of serotonin (5-HT)_1A and/or 5-HT_1B autoreceptors on the fluoxetine-induced increase in dialysate levels of 5-HT as compared with dopamine (DA) and noradrenaline (NAD) in single samples of the frontal cortex (FCx) of freely moving rats. Fluoxetine (10.0 mg/kg, s.c.) elicited a twofold increase in dialysate levels of 5-HT relative to baseline values. The selective 5-HT_1A antagonist WAY 100,635 (0.16 mg/kg, s.c.) did not influence 5-HT release alone but doubled the influence of fluoxetine on basal levels. Similarly, the selective 5-HT_1B/1D antagonist GR 127,935 (2.5 mg/kg, s.c.) did not alter basal 5-HT levels alone and doubled the fluoxetine-induced increase in 5-HT levels. Combined administration of WAY 100,635 and GR 127,935 elicited an (at least) additive rise in the fluoxetine-induced increase in 5-HT levels to eightfold basal values, without modifying resting 5-HT levels. These changes were selective for 5-HT inasmuch as the parallel (twofold) increase in DA and NAD levels provoked by fluoxetine was not potentiated. The present data demonstrate that combined blockade of 5-HT_1A and 5-HT_1B autoreceptors markedly and selectively potentiates the fluoxetine-induced increase in dialysate levels of 5-HT versus DA and NAD in the FCx of freely moving rats. These observations suggest that 5-HT_1A/1B antagonism may represent a novel strategy for the improvement in the therapeutic profile of 5-HT reuptake inhibitor antidepressant agents and that 5-HT may be primarily involved in such interactions.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_1D receptor antagonist N -[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)(1,1-biphenyl)-4-carboxamide (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Regulation of Serotonin Release in the Frontal Cortex and Ventral Hippocampus of Homozygous Mice Lacking 5-HT1B Receptors: In Vivo Microdialysis Studies

Abstract: To assess the involvement of the serotonin receptor subtype 5-HT_1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT_1B receptor. In the frontal cortex and ventral hippocampus, basal and K⁺-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT_1B receptor agonist CP-93,129 (500 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −44%) and ventral hippocampus (by −32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT_1B-5-HT_1D receptor agonist sumatriptan (800 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT_1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT_1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT_1B, but not 5-HT_1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.     

Flesinoxan Dose-Dependently Reduces Extracellular 5-Hydroxytryptamine (5-HT) in Rat Median Raphe and Dorsal Hippocampus Through Activation of 5-HT1A Receptors

Abstract: The effects of systemic administration of the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists flesinoxan and 8-hydroxy-2-(di- n -propylamino)tetralin on extracellular 5-HT were measured using microdialysis probes in both median raphe nucleus and dorsal hippocampus. Both 5-HT_1A agonists dose-dependently decreased dialysate 5-HT levels from both brain regions. The effects of flesinoxan in the median raphe (0.3 mg/kg) and dorsal hippocampus (1.0 mg/kg) could be blocked by the 5-HT_1A receptor antagonist N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexane carboxamide trihydrochloride (WAY 100,635) at a dose of 0.05 mg/kg s.c. The antagonist itself had no effect at this dosage. Local perfusion of flesinoxan for 30 min through the dialysis probe into the median raphe region at concentrations of 20, 100, and 1,000 n M resulted in a significant decrease in dialysate 5-HT content from both regions. The effect of 100 n M flesinoxan could be blocked by coperfusion of 1,000 n M WAY 100,635. The data indicate that flesinoxan is a potent 5-HT_1A receptor agonist and also support the notion that somatodendritic 5-HT_1A autoreceptors regulate both terminal and somatodendritic 5-HT release.     

Guinea pig hippocampal 5-HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT_1E receptor drug development. However, expression of 5-HT_1E receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT_1E drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 – insensitive [³H]5-HT binding (characteristic of the 5-HT_1E receptor). In hippocampal homogenates, where 5-HT_1E receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [³H]5-HT binding at non-5-HT_1E receptors. The K _d of [³H]5-HT was 5.7 ± 0.7 nM and is indistinguishable from the cloned receptor K _d of 6.5 ± 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT_1E receptors are essentially identical ( R ² = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT_1E receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT_1E drugs.     

Characterization of a Mouse Serotonin 5-HT3 Receptor Purified from Mammalian Cells

Abstract: A serotonin 5-HT₃ receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding ∼50% of the histidine-tagged 5-HT₃ receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT₃ receptor is a pentameric homooligomer. The secondary structure of the 5-HT₃ receptor as determined by circular dichroism appeared to consist of mainly α-helices (50%) and β-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.     

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5-hydroxytryptamine (5-HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5-HT receptors. The aim of this study was to subclassify the 5-HT₂ receptor mediating inhibition of the cyclic GMP response elicited by N -methyl- d -aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes, differentially affected the inhibition by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5-HT_2C subtype.