Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

5-HT receptors in mammalian brain: receptor autoradiography andin situ hybridization studies of new ligands and newly identified receptors

Summary In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combinedin situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT_1A receptors in monkey brain, labelled with the agonist³H-8-OH-DPAT and the antagonist³H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT_1A receptors in monkey brain. The labelling conditions to visualize 5-HT_1F receptors in guinea pig brain, namely³H-sumatriptan in the presence of 10⁻⁸ m 5-CT to block 5-HT_1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed byin situ hybridization. By using³H-ketanserin and³H-mesulergine in parallel within situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT_2A and 5-HT_2C binding sites in monkey brain. 5-HT₄ receptors were localized in the brain of several species including humans by using¹²⁵I-SB 207710.In situ hybridization experiments performed in guinea pig confirmed that 5-HT₄ receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT₇ binding sites were labelled in rat and guinea pig brains by incubating with³H-5-CT in the presence of 100 μm WAY 100135 and 250 μm GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Molecular Biology of Serotonin Receptors

Over the last several years the use of molecular cloning technology has revealed a vast diversity among serotonin (5-HT)receptors, where by what was previously thought to be a family of three pharmacologically defined classes of 5-HT receptors is actually composed of seven distinct subfamilies designated 5-HT_1–7. The 5-HT₁, 5-HT₂, and 5-HT₅ subfamilies currently consist of five, three and two subtypes respectively while the 5-HT₃,5-HT₄, 5-HT₆, and 5-HT₇ “subfamilies” have at present one subtype each. Fourteen separate genes encode 13 receptors which fall in the superfamily of G protein-coupled receptors and one ligand-gated ion channel receptor. Our lab has contributed to the elucidation of this subtype diversity by cloning the cDNAs from both rat and human encoding the 5-HT_2B receptor. This receptor subtype is equally homologous (approximately 70%) to the 5-HT_2A and 5-HT_2C receptors when amino acids comprising the transmembrane domains are compared and is clearly the third member of the 5-HT₂ subfamily. The 5-HT_2B receptor has been shown to couple to phosphoinositide hydrolysis as do the other two members of this subfamily when expressed in AV12-664 cells. Limited pharmacological analyses indicated that both rat and human 5-HT_2B receptors are similar but distinguishable. With one tantalizing exception, the mRNAs for these receptors appear to be similarly distributed within rat and human. The 5-HT_2B receptor mRNA is not found in rat brain, whereas in human brain it has been identified in multiple regions. This later finding suggests that the 5-HT_2B receptor may be serving a unique CNS function in man that is absent in rat.     

Relationship between 5-HT2A receptor mRNA density and contractility in trachea and aorta from guinea pig and rat

The present studies document marked differences in contractile responsiveness to serotonin in trachea and aorta between guinea pig and rat. For example, the guinea pig trachea and rat aorta markedly contract in response to serotonin via activation of 5-HT_2A receptors. In contrast, the rat and guinea pig aorta only modestly contract to serotonin. The availability of 5-HT_2A receptor selective cDNA clones from brain of both guinea pig and rat permitted molecular probes to be designed and PCR amplification studies initiated to identify and quantify 5-HT_2A receptor specific mRNA in these tissues. For trachea, 3-fold higher concentrations of 5-HT_2A receptor specific mRNA were found in guinea pig relative to rat trachea. These data are consistent with the more profound contractile response to serotonin in guinea pig versus rat trachea and suggest that differences in tracheal contractility to serotonin correlate with the density of 5-HT_2A receptor mRNA. In contrast, although rat aorta contracted more dramatically to serotonin than guinea pig aorta, rat aorta possessed a similar concentration of 5-HT_2A receptor specific mRNA as compared to guinea pig aorta. Thus, for the aorta, differences in the concentration of 5-HT_2A receptor mRNA are not sufficient to explain the observed differences in contractility between tissues from guinea pig and rat. These studies documenting 5-HT_2A receptor mRNA in rat trachea and guinea pig aorta, two tissues that do not markedly contract in response to serotonin indicate that 5-HT_2A receptor mRNA although present, has not resulted in a receptor capable of mediating a contractile response in these tissues.     

5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release

The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT₃) receptors. We report that recombinantly expressed 5-HT₃ receptor binding sites are reduced by chronic exposure to 5-HT (IC₅₀ of 154.0 ± 45.7 μm, t_½ = 28.6 min). This is confirmed for 5-HT₃ receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC₅₀ of 2.3 ± 1.0 μm, t_½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.     

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.     

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.     

Serotonin 5-HT3 receptors in the central nervous system

The 5-HT₃ receptor is a ligand-gated ion channel activated by serotonin (5-HT). Although originally identified in the peripheral nervous system, the 5-HT₃ receptor is also ubiquitously expressed in the central nervous system. Sites of expression include several brain stem nuclei and higher cortical areas such as the amygdala, hippocampus, and cortex. On the subcellular level, both presynaptic and postsynaptic 5-HT₃ receptors can be found. Presynaptic 5-HT₃ receptors are involved in mediating or modulating neurotransmitter release. Postsynaptic 5-HT₃ receptors are preferentially expressed on interneurons. In view of this specific expression pattern and of the well-established role of 5-HT as a neurotransmitter shaping development, we speculate that 5-HT₃ receptors play a role in the formation and function of cortical circuits.     

Antidepressant-Like Activity of YL-0919: A Novel Combined Selective Serotonin Reuptake Inhibitor and 5-HT1A Receptor Agonist

It has been suggested that drugs combining activities of selective serotonin reuptake inhibitor and 5-HT_1A receptor agonist may form a novel strategy for higher therapeutic efficacy of antidepressant. The present study aimed to examine the pharmacology of YL-0919, a novel synthetic compound with combined high affinity and selectivity for serotonin transporter and 5-HT_1A receptors. We performed in vitro binding and function assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of YL-0919. YL-0919 displayed high affinity in vitro to both 5-HT_1A receptor and 5-HT transporter prepared from rat cortical tissue. It exerted an inhibitory effect on forskolin-stimulated cAMP formation and potently inhibited 5-HT uptake in both rat cortical synaptosomes and recombinant cells. After acute p.o. administration, very low doses of YL-0919 reduced the immobility time in tail suspension test and forced swimming test in mice and rats, with no significant effect on locomotor activity in open field test. Furthermore, WAY-100635 (a selective 5-HT_1A receptor antagonist, 0.3 mg/kg) significantly blocked the effect of YL-0919 in tail suspension test and forced swimming test. In addition, chronic YL-0919 treatment significantly reversed the depressive-like behaviors in chronically stressed rats. These findings suggest that YL-0919, a novel structure compound, exerts dual effect on the serotonergic system, as both 5-HT_1A receptor agonist and 5-HT uptake blocker, showing remarkable antidepressant effects in animal models. Therefore, YL-0919 may be used as a new option for the treatment of major depressive disorder.     

Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain

Recently, a 5-hydroxytryptamine (5-HT) receptor has been described, whose pharmacology was distinct from that of the already known serotonergic receptors, so that it has been called 5-HT₄. Because the lack of a high affinity radioligand, the identification of this receptor depends entirely on functional pharmacological analysis. Its stimulation leads to an increase in cyclic AMP accumulation in mouse embryo colliculi neurons, in guinea pig hippocampus and in human heart. We studied the effect of two indoleamines, 5-HT and 5-methoxytryptamine (5-MeO-T), and a benzimidazolone derivative, BIMU 8, in stimulating basal adenylyl cyclase activity in human frontal cortex, and characterized the receptor subtype involved. In membranes prepared from this tissue, 5-HT, 5-MeO-T and BIMU 8 dose-dependently stimulated (13–25 %) the basal enzyme activity (220 pmoles cyclic AMP/min/mg protein). 5-MeO-T behaved as a full agonist, BIMU 8 elicited about 60 % of the maximal 5-HT effect. The selective 5-HT_1A agonist 8-OH-DPAT, was devoid of any stimulating activity. ICS 205–930, a low affinity 5-HT₄ receptor antagonist, completely reversed the effect of all three agonists at high concentrations. Therefore, the present data are consistent with the 5-HT-mediated stimulation of adenylyl cyclase in human frontal cortex resulting by the activation of a 5-HT₄ receptor subtype.     

SR 57227A is a partial agonist/partial antagonist of 5-HT3 receptor and inhibits subsequent 5-HT- or SR 57227A-induced 5-HT3 receptor current

The serotonin (5-hydroxytryptamine) type 3 (5-HT₃) receptors are transmembrane ligand-gated ion channels. Although several 5-HT₃ receptor agonists have been used as preclinical tools, SR 57227A is the most commonly used 5-HT₃ receptor agonist with the ability to cross the blood brain barrier. However, the precise pharmacological profile of SR 57227A remains unclear. Therefore, we examined the pharmacological profile of SR 57227A at the 5-HT₃A and 5-HT₃AB receptors. We microinjected Xenopus laevis oocytes with human 5-HT₃A complementary RNA (cRNA) or a combination of human 5-HT₃A and human 5-HT₃AB cRNA and performed two electrode voltage clamp recordings of 5-HT₃A and 5-HT₃AB receptor current in the presence of SR 57227A. Results showed that SR 57227A acts as partial agonist/partial antagonist at the 5-HT₃ receptor. Interestingly, SR 57227A specifically reduced subsequent current amplitudes induced by 5-HT or SR 57227A. Based on its 5-HT₃ receptor partial agonist/partial antagonist properties, we predict that SR 57227A functions as a serotonin stabilizer.     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.     

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.     

Agonists and Antagonists Bind to an A-A Interface in the Heteromeric 5-HT3AB Receptor

The 5-HT₃ receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT₃AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT₃AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [³H]granisetron binding affinity (K_d) and 5-HT EC₅₀ were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC₅₀ values were increased. In B-to-A mutant receptors there were no changes in K_d, although EC₅₀ values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT₃A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT₃AB receptor.     

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.     

Pharmacological Characterization of 5-Hydroxytryptamine3Receptors in Murine Brain and Ileum Using the Novel Radioligand [3H]RS-42358–197: Evidence for Receptor Heterogeneity

Abstract: Previous studies have demonstrated species-specific differences in 5-hydroxytryptamine₃ (5-HT₃) receptors, but unequivocal evidence of 5-HT₃ receptor subtypes, within a species, has not yet been obtained. The purpose of the current study was to test for heterogeneity in 5-HT₃ receptors in murine tissues. 5-HT₃ receptors in membranes derived from brain cerebral cortex of CD-1, C57BI/6, and Swiss Webster mice and ileum of CD-1 mice were labeled with the 5-HT₃ receptor antagonist [³H]RS-42358–197. Structurally diverse competing ligands were then used to characterize the binding site. [³H]RS-42358-197 bound with similar affinity in each of the cortical tissues (mean K_D= 0.14 nM; range, 0.06–0.32 nM) but bound with lower affinity in ileal tissue (2.5 nM). The density of sites labeled with [³H]RS-42358–197 ranged from 10.4 fmol/mg of protein in Swiss Webster mouse cortex to 44.2 fmol/mg of protein in Sprague-Dawley rat cortex. Displacing ligands produced a pharmacologic profile of the [³H]RS-42358–197 binding site consistent with it being a 5-HT₃ receptor: (R)-YM060 > (S)-zacopride > (R)-zaco-pride > MDL 72222 > 2-methyl-5-HT. However, 10-fold differences in the affinity of certain ligands were found when comparing 5-HT₃ binding sites in membranes from cerebral cortex of the different strains of mice and when comparing 5-HT₃ binding sites in brain and ileal membranes prepared from the CD-1 mouse strain. Ligands demonstrating selectivity included RS-42358–197, (R)-za-copride, 1-(m-chlorophenyl) biguanide, (R)-YM060, and MDL 72222. These studies demonstrate tissue-and strain-dependent differences in murine 5-HT₃ binding sites. This suggests that 5-HT₃ receptors exist as multiple subtypes within species and that subtype-selective 5-HT₃ ligands may be identified.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

The Central 5-HT3 Receptor in CNS Disorders

Among the characterized 5-HT receptors of the central nervous system, the type 3 receptor subtype (5-HT₃R) is the only one known to be a ligand-gated ion channel. Its early pharmacological characterization and mapping by radioligand binding autoradiography suggested that this receptor may, among other actions, regulate dopamine release in the nigro-striatal pathway and reduce alcohol consumption in experimental animals while antagonists of this receptor have been reported to treat anxiety disorders. Following the cloning of this receptor in 1991, direct cellular localization was made possible by in situ hybridization and immunohistochemical analysis. Here we summarize our recent efforts showing that 5-HT₃R-expressing neurons are mainly GABA containing cells in the rat neocortex, olfactory cortex, hippocampus, and amygdala which also often contain chole-cystokinin (CCK) immunoreactivity. These results provide a means to unify some of the initial pharmacological observations.     

Receptor binding profile of R 41 468, a novel antagonist at 5-HT2 receptors

For a new antiserotonergic agent, R 41 468 and 13 reference compounds with alleged antiserotonergic activity, the receptor binding profile is reported, comprising K_i-values measured in ten different receptor binding models. R 41 468 appeared to be a particularly selective agent with respect to differentiation between two 5-hydroxytryptamine (5-HT) receptor models; it primarily displayed high binding affinity for 5-HT₂ receptors and was inactive at 5-HT₁ receptors. Besides showing a moderate binding affinity for histamine₁ and α₁ adrenergic receptors, the compound was very weakly active at dopamine receptors and inactive at the remaining receptors. Receptor binding profiles of the reference compounds differed widely. Apart from R 41 468 no other compound showed a similar selectivity towards 5-HT₂ receptors. Reference compounds either poorly differentiated between 5-HT₂ and 5-HT₁ receptors, showed other primary effects, or were only moderately active. In the 5-HT₂ and 5-HT₁ receptor binding models the ‘D-receptor’ antagonist phenoxybenzamine was weakly active and the ‘M-receptor’ antagonist morphine was inactive. It is concluded that R 41 468 will be a particularly suitable tool to antagonize 5-HT action mediated by 5-HT₂ receptors.