Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Detection and Characterization of the Serotonin 5-HT1D Receptor in Rat and Human Brain

In the presence of 1 microM ( +/- )-pindolol [to block 5-hydroxytryptamine (5-HT, serotonin) 5-HT 1A and 5-HT 1B receptors] and 100 nM mesulergine (to block 5-HT 1C receptors), 2.0 nM [3H]5-HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5-HT binding, in the presence of 1 microM ( +/- )-pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5-HT receptor subtypes. Compounds with high affinity for 5-HT 1A (8-hydroxydipropylaminotetralin), 5-HT 1B (trifluoromethylphenylpiperazine), 5-HT 1C (mesulergine), 5-HT 2 (4-bromo-2,5-dimethoxyphenylisopropylamine), and 5-HT3 (ICS 205-930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5-HT binding site in rat and human brain tissue. This putative novel 5-HT receptor has a similar pharmacology to the "5-HT 1D" site detected in bovine brain by Heuring and Peroutka.     

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.     

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.     

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.     

Distinct Thermodynamic Parameters of Serotonin 5-HT3 Agonists and Antagonists to Displace [3H]Granisetron Binding

Abstract: Specific binding of [³H]granisetron was examined to serotonin 5-HT₃ receptors in synaptosomal membranes of rat cerebral cortex between 1 and 37°C. Displacing potencies were determined for 5-HT₃ antagonists (granisetron, ondansetron, tropisetron, and d -tubocurarine) and agonists (5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, phenylbiguanide, m -chlorophenylbiguanide, and SR 57227A). Displacing potencies of the agonists decreased with decreasing temperature. In contrast, displacing potencies of all antagonists increased with decreasing temperature, whereas those of tropisetron and d -tubocurarine passed a maximum. Scatchard analysis of [³H]granisetron binding resulted in K _D values lower than the IC₅₀ values of granisetron and a decreasing number of binding sites at higher temperatures. It can be reconciled with temperature-dependent agonist and antagonist states of 5-HT₃ receptors. A semiquantitative thermodynamic analysis was based on displacing potencies. The distinct patterns for the signs of entropy, enthalpy, and heat capacity changes on binding can be reconciled with ionic interactions for agonists and hydrophobic interactions for antagonists. The distinctive differences in these thermodynamic parameters exceed those for GABA_A and glycine receptor-ionophore complexes.     

Two Amino Acid Differences in the Sixth Transmembrane Domain Are Partially Responsible for the Pharmacological Differences Between the 5-HT1Dβ and 5-HT1E 5-Hydroxytryptamine Receptors

Abstract: 5-Hydroxytryptamine elicits its physiological effects by interacting with a diverse group of receptors. Two of these receptors, the 5-HT_1Dβ and the 5-HT_1E receptors, are ∼60% identical in the transmembrane domains that presumably form the ligand binding site yet have very different pharmacological properties. Analysis of the pharmacological properties of a series of chimeric 5-HT_1Dβ/5-HT_1E receptors indicates that sequences in the sixth and seventh transmembrane domains are responsible for the differential affinity of 5-carboxamidotryptamine for these two receptors. More detailed analysis shows that two amino acid differences in the sixth transmembrane domain (Ile³³³ and Ser³³⁴ in the 5-HT_1Dβ receptor, corresponding to Lys³¹⁰ and Glu³¹¹ in the 5-HT_1E receptor) are largely responsible for the differential affinities of some, but not all, ligands for the 5-HT_1Dβ and 5-HT_1E receptors. It is likely that these two amino acids subtly determine the overall three-dimensional structure of the receptor rather than interact directly with individual ligands.     

Serotonin 5-HT2C Receptor RNA Editing Alters Receptor Basal Activity

Rat and human serotonin 5-HT2C receptor isoforms were evaluated for agonist-independent activation of inositol phosphate production in COS-7 cells. The nonedited isoform (5-HT(2C-INI)) displayed the greatest basal activity, stimulating inositol phosphate production fourfold over the fully edited isoform (5-HT(2C--VGV)). All of the other isoforms tested displayed intermediate levels of basal activity. Decreasing receptor expression levels by 50% produced a parallel decrease in basal activity. 5-HT stimulated inositol phosphate production twofold over basal levels through the 5-HT(2C-INI) receptor and eightfold over basal levels through the 5-HT(2C-VGV) receptor but produced similar maximal levels of inositol phosphate. 5-HT competition for [3H]mesulergine binding to 5-HT(2C-INI) best fit a two-site analysis with K(H) = 7.6 nM and K(L) = 160 nM, whereas 5-HT(2C-VGV) best fit a one-site model with Ki = 163 nM. [3H]5-HT labeled 36% of the total population of 5-HT(2C-INI) receptors labeled by [3H]mesulergine but only 12% of 5-HT(2C-VGV) receptors. [H]5-HT K(D) values increased from 5.1 nM for 5-HT(2C-INI) to 20 nM for 5-HT(2C-VGV). [3H]Mesulergine K(D) values were the same for both isoforms. 5-HT EC50 values for inositol phosphate production increased from 6.1 nM for 5-HT(2C-INI) to 30 nM for 5-HT(2C-VGV). These results demonstrate that RNA editing decreases 5-HT2C receptor basal activity, agonist affinity, and potency, indicating that RNA editing may play a role in regulating serotonergic signal transduction and response to drug therapy.     

Primary Structure of the Human Platelet Serotonin 5-HT2A Receptor: Identity with Frontal Cortex Serotonin 5-HT2A Receptor

Abstract: Previous radioligand binding studies have demonstrated human platelet serotonin_2A (5-HT_2A) receptor binding sites. Pharmacological similarities between platelet and frontal cortex 5-HT_2A receptor binding parameters have been demonstrated. However, it is not clear whether the platelet 5-HT_2A receptor primary structure is identical to that of the brain receptor. Three overlapping cDNAs were obtained to span completely the coding region of the 5-HT_2A receptor. These clones were sequenced with external and internal primers. The nucleotide sequence of human platelet 5-HT_2A cDNA was identical to that reported for the human frontal cortex 5-HT_2A receptor, except for nucleotide 102 (T → C), which has been reported to represent a normal DNA polymorphism that does not alter the amino acid sequence. This finding may have implications in the study of neuropsychiatric disorders for which altered platelet 5-HT_2A receptor binding has been demonstrated.     

Native Serotonin 5-HT3 Receptors Expressed in Xenopus Oocytes Differ from Homopentameric 5-HT3 Receptors

Abstract: Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT₃ receptor (5-HT₃R) agonists 2-methyl-5-HT, dopamine, and m -chlorophenylbiguanide on 5-HT₃R native to N1E-115 cells and on homopentameric 5-HT₃R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT₃R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT₃R-A_L and 5-HT₃R-A_s receptors expressed in oocytes (4–8%). m -Chlorophenylbiguanide does not distinguish between 5-HT₃R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT₃R native to differentiated N1E-115 cells is conserved when poly(A)⁺ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT₃R subunits, N1E-115 cells express additional proteins involved in 5-HT₃R function.     

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.     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.     

Detection of a Novel Serotonin Receptor Subtype (5-HT1E) in Human Brain: Interaction with a GTP-Binding Protein

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acquisition of and withdrawal from cocaine self-administration regulates 5-HT1B mRNA expression in rat striatum

This study investigated how different stages of cocaine self-administration in rats affect the expression of two serotonin receptors in dorsal and ventral striatum, the 5-HT_1B and 5-HT₆ subtypes, which have both been implicated in mediating some aspects of cocaine-related behaviors. In the first experiment, rats were trained to work for saccharin (oral) or cocaine (i.v.) reinforcers. We found that continuous access to cocaine for 23 days did not change the level of 5-HT_1B mRNA expression compared to control animals receiving saccharin. However, a single cocaine session, given either by self-administration or non-contingently, increased 5-HT_1B mRNA in dorsal striatum, whereas forced abstinence for two weeks after cocaine reduced 5-HT_1B mRNA expression in the same subregion. 5-HT₆ mRNA was not changed by any of these treatments. A follow-up experiment investigated the effects of limited versus extended access to cocaine as well as forced abstinence, and we found that 14 days of forced abstinence significantly reduced 5-HT_1B mRNA throughout the dorsal and ventral striatum compared to no withdrawal. These results suggest that the influence of 5-HT_1B receptors in striatal projection neurons may be increased during cocaine acquisition and reduced after forced abstinence and may therefore be targets for pharmacological intervention in addiction.     

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.     

[3H]Spiroxatrine: A 5-HT1A Radioligand with Agonist Binding Properties

Spiroxatrine has been reported to be a 5-HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5-HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5-HT1A antagonist radioligand. [3H]8-Hydroxydipropylaminotetralin [( 3H]8-OH-DPAT), a well-characterized 5-HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8-OH-DPAT binding produced KD values of 0.9 nM and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r = 0.98; p less than 0.001) exists between Ki values obtained for a series of drugs in competing for [3H]-spiroxatrine and [3H]8-OH-DPAT binding. Of special interest was the observation that 5-HT1A agonists such as serotonin, 8-OH-DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8-OH-DPAT-labelled 5-HT1A receptors. [3H]Spiroxatrine and [3H]8-OH-DPAT binding to 5-HT1A receptors was inhibited by guanosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration-dependent manner whereas adenosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of ATP) had no effect. The similarities in the 5-HT1A receptor radiolabelling properties of [3H]spiroxatrine and [3H]8-OH-DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has "agonist-like" binding properties in its interaction with the 5-HT1A receptor.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these 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.     

Differential Coupling of Serotonin 5-HT1A and 5-HT1B Receptors to Activation of ERK2 and Inhibition of Adenylyl Cyclase in Transfected CHO Cells

Although the subtypes of serotonin 5-HT1 receptors have distinct structure and pharmacology, it has not been clear if they also exhibit differences in coupling to cellular signals. We have sought to compare directly the coupling of 5-HT1A and 5-HT1B receptors to adenylyl cyclase and to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase-2). We found that 5-HT1B receptors couple better to activation of ERK2 and inhibition of adenylyl cyclase than do 5-HT1A receptors. 5-HT stimulated a maximal fourfold increase in ERK2 activity in nontransfected cells that express endogenous 5-HT1B receptors at a very low density and a maximal 13-fold increase in transfected cells expressing 230 fmol of 5-HT1B receptor/mg of membrane protein. In contrast, activation of 5-HT1A receptors stimulated only a 2.8-fold maximal activation of ERK2 in transfected cells expressing receptors at 300 fmol/mg of membrane protein but did stimulate a 12-fold increase in activity in cells expressing receptors at 3,000 fmol/mg of membrane protein. Similarly, 5-HT1A, but not 5-HT1B, receptors were found to cause significant inhibition of forskolin-stimulated cyclic AMP accumulation only when expressed at high densities. These findings demonstrate that although both 5-HT1A and 5-HT1B receptors have been shown to couple to G proteins of the Gi class, they exhibit differences in coupling to ERK2 and adenylyl cyclase.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.