Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

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.     

Ultrastructure of the 5-Hydroxytryptamine3 Receptor

Abstract: We have determined the ultrastructure of 5-hydroxytryptamine₃ (5-HT₃) serotonin receptors purified from NG108-15 mouse neuroblastoma × rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT₃ receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT₃ receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.     

Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging

Abstract: 5-Hydroxytryptamine₃ (5-HT₃) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABA_A, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT₃ receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT₃ receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT₃ serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels.     

A Single Amino Acid Difference Accounts for the Pharmacological Distinctions Between the Rat and Human 5-Hydroxytryptamine1B Receptors

Abstract: Molecular cloning of the rat and human 5-hydroxytryptamine_1B (5-HT_1B) receptors has revealed that the primary amino acid sequence of these two receptors is >90% identical. Despite this high degree of primary sequence homology, these two receptors have significantly different pharmacological properties. A mutant human 5-HT_1B receptor was constructed in which Thr³⁵⁵ was replaced by Asn, the corresponding residue at this position in the rat 5-HT_1B receptor. The pharmacology of the mutant human 5-HT_1B receptor was very similar to that of the rat 5-HT_1B receptor. Specifically, the mutant receptor had much higher affinity for pindolol, [¹²⁵I]-iodocyanopindolol, propranolol, and CP-93,129 than the wild-type receptor. In contrast, the mutant had significantly lower affinity for sumatriptan, N,N -dipropyl-5-carboxamidotryptamine, 5-methoxy- N,N -dimethyltryptamine, methysergide, metergoline, and rauwolscine. These data suggest that a single amino acid difference at position 355 is responsible for the pharmacological differences between the rat and human 5-HT_1B receptors.     

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.     

Identification and Characterisation of 5-Hydroxytryptamine3 Recognition Sites in Human Brain Tissue

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.     

5-Hydroxytryptamine3 Receptors Sited on Cholinergic Axon Terminals of Human Cerebral Cortex Mediate Inhibition of Acetylcholine Release

Synaptosomes prepared from freshly obtained human cerebral cortex and labeled with [3H]choline have been used to investigate the modulation of [3H]acetylcholine ([3H]ACh) release by 5-hydroxytryptamine (5-HT). The Ca(2+)-dependent release of [3H]-ACh occurring when synaptosomes were exposed in superfusion to 15 mM KCl was inhibited by 5-HT (0.01-1 microM) in a concentration-dependent manner. The effect of 5-HT was mimicked by 1-phenylbiguanide, a 5-HT3 receptor agonist, but not by 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A receptor agonist. The 5-HT3 receptor antagonists tropisetron and ondansetron blocked the effect of 5-HT, whereas spiperone and ketanserin were ineffective. It is suggested that cholinergic axon terminals in the human cerebral cortex possess 5-HT receptors that mediate inhibition of ACh release and appear to belong to the 5-HT3 type.     

Antisense Oligonucleotide-Induced Reduction in 5-Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function

Abstract: The effect of a 5-hydroxytryptamine₇ (5-HT₇) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT₇ antisense oligonucleotide treatment significantly reduced [³H]5-HT binding to hypothalamic 5-HT₇ receptors, whereas cortical 5-HT_2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT₇ antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT₇ antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT₇ antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT₇ receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT₇ receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.     

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.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

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.     

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.     

Barbiturates Are Selective Antagonists at A1 Adenosine Receptors

Barbiturates in pharmacologically relevant concentrations inhibit binding of (R)-N6-phenylisopropyl[3H]adenosine ([3H]PIA) to solubilized A1 adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. Ki values are similar to those obtained for membrane-bound receptors and are 31 microM for (+/-)-5-(1,3-dimethyl)-5-ethylbarbituric acid [(+/-)-DMBB] and 89 microM for (+/-)-pentobarbital. Kinetic experiments demonstrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-N6-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The stimulation of adenylate cyclase via A2 adenosine receptors in membranes from N1E 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. It is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A1 adenosine receptor antagonism may convey excitatory properties to barbiturates.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

5-Hydroxytryptamine2 and β-Adrenergic Receptor Regulation in Rat Brain Following Chronic Treatment with Desipramine and Fluoxetine Alone and in Combination

Abstract: A chronic (14-day) study was initiated to investigate the effects of combined fluoxetine (FLU) and desipramine (DMI) treatment on the densities and affinities of β-adrenergic and 5-hydroxytryptamine₂ (5-HT₂) receptors. Male Sprague-Dawley rats were administered the following doses using osmotic minipumps: FLU, 10 mg/kg/day; DMI, 5, 10, or 15 mg/kg/day; FLU, 10 mg/kg/day, plus DMI, 5 mg/kg/day; or vehicle (distilled water). After 14 days the cortex was dissected out and used for [³H]-ketanserin (5-HT₂) binding, [³H]CGP-12177 (β-adrenergic) binding, and drug level analysis. All animals receiving DMI showed significant down-regulation of 5-HT₂ receptors except those receiving FLU in combination. DMI down-regulated β-adrenergic receptors in a dose-dependent manner, with significantly greater down-regulation seen with the combination than with DMI (5 mg/kg/day) alone. This latter effect was apparently the result of greater levels of DMI in cortex with the combination than with DMI (5 mg/kg/day) alone. FLU had no effect on 5-HT₂ or β-adrenergic receptors on its own. Coadministration of FLU and DMI resulted in a doubling of levels of FLU and its demethylated metabolite, norfluoxetine (NFLU), and a tripling of DMI levels compared with values observed when FLU (10 mg/kg/day) or DMI (5 mg/kg/day) was administered alone. These results suggest that with the DMI/FLU combination (a) FLU and/or NFLU block the down-regulation of 5-HT₂ receptors caused by DMI alone, (b) an important factor determining β-adrenergic receptor density may be the elevated DMI levels relative to those with DMI (5 mg/kg/day) alone, (c) FLU and/or NFLU inhibit the metabolism of DMI, and (d) DMI inhibits the metabolism of FLU.