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5-HT receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-HT receptors [194] and subsequently revised [176]) are, with the exception of the ionotropic 5-HT3 class, GPCRs where the endogenous agonist is 5-hydroxytryptamine. The diversity of metabotropic 5-HT receptors is increased by alternative splicing that produces isoforms of the 5-HT2A (non-functional), 5-HT2C (non-functional), 5-HT4, 5-HT6 (non-functional) and 5-HT7 receptors. Unique amongst the GPCRs, RNA editing produces 5-HT2C receptor isoforms that differ in function, such as efficiency and specificity of coupling to Gq/11 and also pharmacology [40, 482]. Most 5-HT receptors (except 5-ht1e and 5-ht5b) play specific roles mediating functional responses in different tissues (reviewed by [463, 382]).

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.

Screening technologies for G protein-coupled receptors comprise two major approaches; homogeneous assays, conducted in microtiter plate formats, and protein redistribution assays that require imaged-based analysis using automated confocal systems. Generally, the former are used in primary screening campaigns for lead identification, while the latter are used in secondary screens for lead optimization. Homogeneous assays measure changes in G protein-coupled receptor second messengers such as cAMP, Ins P3 or calcium. Protein redistribution assays assess internalization of the receptor ligand complex or translocation of G-protein coupled receptor-associated proteins, such as beta-arrestin. In the present review functional cell based approaches are discussed, emphasizing the variety of non radiometric technologies now in use for HTS.

G-protein coupled receptors (GPCRs) represent the most tractable family of drug targets. Those GPCRs identified by sequence only, but lacking an endogenous ligand, are defined as orphan GPCRs (oGPCRs) and might represent the next generation of targets for GPCR drug discovery. Drug discovery at oGPCRs is a resource intensive approach and frequently taken 'at-risk' without a clear understanding of the role in a disease. Identification of oGPCRs is, therefore, a prerequisite for the initiation of a drug discovery program.

Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.

A cDNA clone (designated as GP2-7) encoding a novel 5-hydroxytryptamine (5-HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5-HT receptor subtypes (34–48%). The sequence of GP2-7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5-HT7. mRNA for GP2-7 was detected in cortical and limbic brain regions. Transiently expressed GP2-7 showed high-affinity binding to [3H]5-HT (pKi = 9.0) with the following rank order of affinities: 5-carboxyamidotryptamine (5-CT) > 5-HT = 5-methoxytryptamine (5-MeOT) > methiothepin > 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) > spiperone ≫ sumatriptan. Adenylyl cyclase activity in CHO-K1 cells transiently transfected with GP2-7 was stimulated by several analogues of 5-HT with the following order of potency: 5-CT > 5-HT = 5-MeOT > dipropyl-5-CT > 8-OH-DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2-7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5-CT.

Muscarinic cholinoceptors (MChR) in freshly dispersed rat salivary gland (RSG) cells were characterized using microphysiometry to measure changes in acidification rates. Several non-selective and selective muscarinic antagonists were used to elucidate the nature of the subtypes mediating the response to carbachol. The effects of carbachol (pEC50=5.74±0.02 s.e.mean; n=53) were highly reproducible and most antagonists acted in a surmountable, reversible fashion. The following antagonist rank order, with apparent affinity constants in parentheses, was noted: 4-DAMP (8.9)=atropine (8.9)>tolterodine (8.5)>oxybutynin (7.9)>S-secoverine (7.2)>pirenzepine (6.9)>himbacine (6.8)>AQ-RA 741 (6.6)>methoctramine (5.9). These studies validate the use of primary isolated RSG cells in microphysiometry for pharmacological analysis. These data are consistent with, and extend, previous studies using alternative functional methods, which reported a lack of differential receptor pharmacology between bladder and salivary gland tissue. The antagonist affinity profile significantly correlated with the profile at human recombinant muscarinic M3 and M5 receptors. Given a lack of antagonists that discriminate between M3 and M5, definitive conclusion of which subtype(s) is present within RSG cells cannot be determined. British Journal of Pharmacology (2001) 132, 1606–1614; doi:10.1038/sj.bjp.0703971

Over the last decade, several lines of evidence have shown that both muscarinic M2 and M3 receptors are postjunctionally expressed in many smooth muscles, including the gastrointestinal tract. Although in vitro data suggests that both receptors are functional in that they inhibit adenylate cyclase activity and activate non-selective cation channels, few studies support a role in vivo. Thus, data from procedures that ablate the signaling pathway of the muscarinic M2 receptor, including receptor antagonism, pertussis toxin pretreatment reveal little effect on gastrointestinal smooth muscle responsiveness to muscarinic agonists. Recently, information from knockout mice, lacking either M2 or M3 receptor, indicate reveal a role for both subtypes. However, the contribution of the M2 receptor appears greater in the ileum than in the urinary bladder. Therapeutically, non-selective, as well as selective M3 receptor antagonists are being clinically studied, although it remains to be shown which is the optimal approach to disorders of smooth muscle motility.

The muscarinic acetylcholine receptors belong to the super-family of seven TM domain receptors that interact with G-proteins to initiate intracellular responses. Evidence from molecular cloning indicates that there are separate intronless human genes that encode five muscarinic receptor glycoproteins. Muscarinic receptor sequences have significant homologies with other members of this large super-family and the genes are very similar across mammalian species (Caulfield 1993; Felder 1995). Despite over a decade following their molecular identification, the therapeutic exploitation of this crucial family of receptors remains disappointing. This results from a relative inability to pharmacologically distinguish between the subtypes, markedly hindering their investigation in native mammalian tissues. In particular, this has hampered investigation of the last-identified subtype, the muscarinic M5 receptor. The inability to clearly distinguish it from the M3 receptor has, moreover, led to confusion of its physiological role. Finally, determination of the precise distribution of M5 receptors within tissues is complicated by inadequate selectivity of radioligands as well as the low sensitivity/selectivity of polyclonal antisera in immunocytochemical studies (Caulfield 1993; Reever et al., 1997). Despite these problems, this receptor has recently been assigned an upper case M5 nomenclature (Caulfield & Birdsall, 1998) presumably reflecting recognition of its presence and function in native tissues despite the current incomplete characterization. In this respect the identification of a human A2058 melanoma cell that endogenously expresses the M5 receptor (Kohn et al., 1996) should facilitate its investigation in endogenous tissues, although extensive use of these cells have not been reported to date. Consequently, the majority of the current information on the functional properties and regulation of coupling of this subtype still arises from their expression in model cells following cDNA transfection. The purpose of this short review is to critically evaluate current data on the muscarinic receptor M5 subtype from several standpoints. Hopefully, this critique will stimulate further studies on the M5 receptor that may raise it from a 'relatively ephemeral' or 'fact or fiction' status, described in recent reviews (Reever et al., 1997; Caulfield & Birdsall 1998). Identification and structural features The muscarinic M5 receptor was the last of the muscarinic receptor family to be cloned in the human and is mapped to chromosome 15q26 (Bonner et al., 1988; Liao et al., 1989). The receptor sequence conforms to a predicted seven transmembrane glycoprotein consisting of 531 residues in the human (GeneBank accession number PO8912) and 532 in the mouse (PO8911; 89% homologous to human). Structurally, the M5 receptor is the next largest muscarinic receptor to the M3 subtype with both these subtypes possessing a large third intracellular loop. Differences in this cytoplasmic loop account for the sequence diversity between muscarinic receptor subtypes and also between muscarinic receptors from different species. However, of the five muscarinic receptors, the M5 subtype demonstrates the least homology in this region when comparisons are made between human and rat sequences. Wess and colleagues (Wess et al., 1992; Pittel & Wess, 1994; Wess, 1997) have explored the nature of ligand binding and G-protein coupling by using chimeras of muscarinic M2 and M5 receptors. Most M2/M5 constructs are inactive but the presence of the M2 sequence in TMVII and M5 in TMI agonist activation of G-protein coupling is restored. Pittel & Wess (1994) argued that these data supported the bacteriorhodopsin model in which the seven transmembrane helices are arranged in a ring, such that TMI is adjacent to TMVII. A series of M2/M5 chimeras in which regions of the M5 receptors have been systemically replaced by homologous regions of the M2 receptor, indicated the higher affinity of the antagonist UH-AH 37 for the M2 over the M5 receptor was dependent upon a short stretch of 31 residues in TMVI as well as a short region of the third intracellular loop. This however contrasts to the antagonist AQ-RA 741 which also preferentially binds to the M2 receptor suggesting that different receptor epitopes may be involved in conferring different ligand specificities. In a series of studies, Brann and colleagues also attempted to identify key residues associated with agonist activation of M5 receptors. Initially using random saturation mutagenesis they identified the amino acids 439, A440, A441 towards the C-terminal end of the third intracellular loop of the M5 muscarinic receptor critical for G-protein coupling (Burstein et al., 1995). In a more recent paper, this group (Burstein et al., 1998a) constructed a further series of point mutants at each of these residues and characterized their functional phenotypes in order to find structure function relationship for G-protein coupling to the M5 receptor. Their evidence suggests that residue 439 participates in G-protein activation through an ionic mechanism and that A440 fulfils more of a structural role, perhaps forming part of the G-protein coupling pocket. Further, A441 apparently contributes to receptor affinity for G-proteins. Collectively, these data suggest that the third intracellular loop of the M5 receptor forms a G-protein coupling pocket comprised of a positively charged lip and a hydrophobic core. Brann's group (Spalding et al., 1998) also investigated a potential switch between active and inactive conformations of the M5 muscarinic receptor. There is much evidence from several G-protein coupled receptors to suggest that G-protein receptors exhibit constitutive activity (i.e. activation in the absence of agonist) and that agonists stabilize active whereas antagonists stabilise inactive conformations (Kenakin 1996; 1997). In a search for residues that participate in receptor function, several regions of the M5 receptor were randomly mutated and tested for their functional properties. Mutations spanning the face of TMVI were found to induce high levels of constitutive activity of the receptor. The same face of TMVI contained several residues crucial to receptor activation by agonists and one residue was identified as a contact site for both agonists and antagonists. These results suggest that within TMVI of the M5 receptor is a switch that defines the activation state of the receptor and the ligand interactions with TMVI stabilizing the receptor in either active or inactive conformations. In a further study (Burstein et al., 1998b) this group completed a systematic search of the intracellular loops in an attempt to identify further domains that govern G-protein coupling. A feature of the second intracellular loop was an ordered cluster of residues where substitutions also cause constitutive activation of the M5 receptor. A second group of residues in the second intracellular loop have been identified where mutations compromise receptor/G-protein coupling. The residues of each group appear to alternate and are spaced three to four positions apart, perhaps suggesting an α-helical structure where the groups form opposing faces of the helix. The authors suggest that the constitutively activating face normally constrain the receptor in the off state while the other face couples to G-proteins with the receptor being in the on state.

Human α2A-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca2+i) changes using the Ca2+-sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). Several α-adrenoceptor agonists were examined including the α2-adrenoceptor agonists UK-14304, B-HT 920, dexmedetomidine and A-54741, the selective α1-adrenoceptor agonist phenylephrine and the non-selective adrenergic agonist noradrenaline. Of these only noradrenaline (mean pEC50=6.49) and A-54741 (6.90) evoked changes in Ca2+i; A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. Ca2+i changes induced by noradrenaline and A-54741 were antagonized by the α2-selective antagonist rauwolscine (10 nM) and by the α1-selective antagonists prazosin (0.1 nM) and doxazosin (1.0 nM). Phenylephrine (100 μM) and UK-14304 (10 μM) alone were ineffective in causing Ca2+i increase. In the presence of a fixed concentration of UK-14304 (3.0 μM), phenylephrine induced concentration-dependent increases in Ca2+i (mean pEC50=5.33). In the presence of phenylephrine (30.0 μM) UK-14304 induced Ca2+i release (pEC50=6.92). The effects of phenylephrine were abolished by prazosin (1.0 nM) or rauwolscine (100 nM). In saturation radioligand binding experiments using membranes of parental (non-transfected) CHL cells there was a small, specific binding of [3H]-prazosin (Bmax=24 fmol mg protein−1; pKD=10.24). Collectively, these data suggest that α-adrenoceptor agonist-induced Ca2+i release in CHL fibroblasts transfected with the human α2A-adrenoceptor is dependent upon co-activation of the recombinant receptor and a native α1-adrenoceptor. British Journal of Pharmacology (2000) 129, 1339–1346; doi:10.1038/sj.bjp.0703184

Over the past year, the introduction of novel ligands has accelerated the classification of muscarinic receptor subtypes and has led to a better understanding of their physiological role. Important in this respect is the recent recognition of the exquisite selectivity of a series of snake toxins, enabling better definition of the muscarinic subtype 4 receptor. Moreover, several compounds, both agonists and antagonists, are progressing in advanced clinical trials for the treatment of several conditions, including Alzheimer's disease, pain, urinary incontinence and chronic obstructive pulmonary disease.

Efforts to define precisely the role of 5-HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5-HT2B receptor antagonists. RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar affinity for the 5-HT2B receptor (pKi=9.5±0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. In cells expressing human recombinant 5-HT2B receptors, RS-127445 potently antagonized 5-HT-evoked formation of inositol phosphates (pKB=9.5±0.1) and 5-HT-evoked increases in intracellular calcium (pIC50=10.4±0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA2=9.5±1.1) and (±)α-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2=9.9±0.3). RS-127445 had no detectable intrinsic activity in these assays. In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg−1) produced plasma concentrations predicted to fully saturate accessible 5-HT2B receptors for at least 4 h. In conclusion, RS-127445 is a selective, high affinity 5-HT2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated. British Journal of Pharmacology (1999) 127, 1075–1082; doi:10.1038/sj.bjp.0702632

Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. For the three alpha 2-adrenergic receptors (alpha 2A, alpha 2B, and alpha 2C), genetic mouse models have been developed that can be used to elucidate the physiologic function of each receptor subtype in vivo. Different strategies for homologous recombination in embryonic stem cells were applied to generate lines of mice with gene knockouts of the individual alpha 2-receptor subtypes (alpha 2A-KO, alpha 2B-KO, and alpha 2C-KO) or with a substitution of a mutant receptor at the wild-type locus (alpha 2-D79N). In these transgenic mice, the cardiovascular effects of alpha 2-agonists and imidazoline receptor agonists were tested. Stimulation of alpha 2B receptors in vascular smooth muscle produces hypertension and counteracts the clinically beneficial hypotensive effect of stimulating alpha 2A receptors in the central nervous system.

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the alpha1A-adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the alpha1-adrenoceptor (alpha1-AR) subtype selectivities of two novel alpha1-AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned alpha1A-, alpha1B- and alpha1D-AR showed nanomolar affinity and significant alpha1A-AR subtype selectivity for both Ro 70-0004 (pKi 8.9: 60 and 50 fold selectivity) and RS-100329 (pKi 9.6: 126 and 50 fold selectivity) over the alpha1B- and alpha1D-AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA2 8.8 and 8.9), RS-100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing alpha1-AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The alpha1A-AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a 'uroselective' agent for the treatment of symptoms associated with benign prostatic hyperplasia.

1. Affinity estimates were obtained for several muscarinic antagonists against carbachol-stimulated [3H]-inositol phosphates accumulation in Chinese hamster ovary (CHO-KI) cells stably expressing either human muscarinic M3 or M5 receptor subtypes. The rationale for these studies was to generate a functional antagonist affinity profile for the M5 receptor subtype and compare this with that of the M3 receptor, in order to identify compounds which discriminate between these two subtypes. 2. The rank order of antagonist apparent affinities (pK(B)) at the muscarinic M5 receptor was atropine (8.7) > or =tolterodine (8.6) = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.6)> darifenacin (7.7) > or =zamifenacin (7.6)>oxybutynin (6.6)= para-fluorohexahydrosiladifenidol (p-F-HHSiD, 6.6)>pirenzepine (6.4) > or = methoctramine (6.3)=himbacine (6.3)>AQ-RA 741 (6.1). 3. Antagonist apparent affinities for both receptor subtypes compare well with published binding affinity estimates. No antagonist displayed greater selectivity for the muscarinic M5 subtype over the M3 subtype, but himbacine, AQ-RA 741, p-F-HHSiD, darifenacin and oxybutynin displayed between 9- and 60 fold greater selectivity for the muscarinic M3 over the M5 subtype. 4. This study highlights the similarity in pharmacological profiles of M3 and M5 receptor subtypes and identifies five antagonists that may represent useful tools for discriminating between these two subtypes. Collectively, these data show that in the absence of a high affinity M5 selective antagonist, affinity data for a large range of antagonists is critical to define operationally the M5 receptor subtype.

In many smooth muscle tissues a minor M3-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M3-mACh receptors. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. Antagonist affinity estimates made at 'protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine> methoctramine. Carbachol-induced contractions were insensitive to the M4-selective muscarinic toxin MTx-3, or PD102807 (0.1 microM) ruling out a role for M4-mACh receptors. The agonist affinity value for L-660,863, a putative 'M2-selective' agonist of 5.44+/-0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pKA value for carbachol (4.22+/-0.17 n = 8) agrees with that reported for guinea-pig ileum. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi-proteins in contraction. Radioligand binding studies revealed a KD for N-[3H]-methylscopolamine of 0.12+/-0.05 nM and a Bmax of 147+/-18 fmol mg protein(-1). Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2-mACh receptors. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

1. Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG x neuroblastoma hybrid) cell line. 2. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2alpha had negligible effects. The rank order of agonist potency was cicaprost>PGE2=BMY45778=cPGI2=PGI2. In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2++ +. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. 3. To examine the effects of prostanoids on C-fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the 'grease-gap' technique. PGI2 (0.1 nM-10 microM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2alpha. 4. Prior depolarization of nerves with either forskolin (10 microM) or phorbol dibutyrate (1 microM) alone significantly reduced the response to PGI2 (10 microM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. 5. Putative EP1 and/or TP receptor-selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. 6. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.

Alpha-2 adrenergic receptors (α2 AR) mediate incorporation of guanosine 5′-O-(γ-thio)triphosphate ([35S]GTPγS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPγS for GDP. In the current study, we used [35S]GTPγS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse α2a/d and human α2a, α2b, and α2c ARs. Full agonists increased [35S]GTPγS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPγS binding but competitively inhibited agonist-stimulated [35S]GTPγS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPγS binding. The agonistic properties of a number of α2 AR ligands were characterized at each α2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: α2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). α2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). α2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) ≥ clonidine (0.23) ≫ guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse α2a/d AR. It also illustrates the utility of [35S]GTPγS incorporation as a functional marker of receptor activation.

Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the generation of hyperalgesia. It is generally thought that prostaglandin (PG) E2 is the principal pro-inflammatory prostanoid. Consequently, prostanoid EP receptors on sensory neurones have been identified as potential therapeutic targets. However, IP prostanoid receptors are also present on sensory neurones, and recent data from transgenic mice lacking the IP receptor demonstrate its importance in the induction of oedema and pain behaviour. PGI2, the primary endogenous agonist for the IP receptor, is rapidly produced following tissue injury or inflammation; thus, it may be of equal, or greater, importance than PGE2 during episodes of inflammatory pain. In this review, Keith Bley, John Hunter, Richard Eglen and Jacqueline Smith compare the roles of EP and IP receptors in nociception and suggest that the IP receptor constitutes a novel target for anti-nociceptive agents.

Recent evidence indicates that the 5-HT4 subtype of serotonin receptor may modulate central cholinergic activity in regions of the mammalian CNS important to memory such as the frontal cortex, hippocampus and amygdala. These receptors could represent targets for drugs designed for the symptomatic therapy of Alzheimer's disease (AD) and other disorders of memory. In the present study, the binding activity of RS 17017 (previously described as a selective 5-HT4 agonist) was assessed across a number of neurotransmitter receptors and binding sites, pharmacokinetic data were obtained, and the compound was evaluated in macaques for mnemonic effects via a computer-assisted delayed matching-to-sample task (DMTS). Binding data confirmed the 5-HT4 selectivity of the compound, while pharmacokinetic results revealed low oral bioavailability, but a large volume of distribution of the compound. Significant and reproducible improvements in DMTS accuracy were observed after oral administration of the compound across a dose-effect series in both younger and older monkeys. The results suggest that RS 17017 offers a potential for memory enhancement in disorders involving cognitive decline, and are consistent with a role for central 5-HT4 receptors in memory. Improvements in DMTS performance in aged monkeys may have particular implications for neurodegenerative conditions such as AD, whereas positive results in the younger monkeys indicate that RS 17017 (or similar compounds) may have additional potential in the therapeutics of memory disorders not necessarily associated with advanced age.

1 The rat 5-hydroxytryptamine (5-HT)7 receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT7 receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor. 2 A truncated splice variation in the human 5-HT7 receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT7(b) receptor and the long form of the receptor as h5-HT7(a). 3 The h5-HT7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [3H]-5-carboxyamidotryptamine (5-CT; Kd=0.28±0.06 nM, Bmax=7.3±1.7 pmol mg−1 protein). The rank order of affinities (pKi) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44). 4 The h5-HT7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC50): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies. 5 In a similar fashion to that described for the 5-HT7(a) receptor, PCR studies suggested that the 5-HT7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta. 6 It is concluded that the h5-HT7 receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT7 receptor gene. British Journal of Pharmacology (1997) 122, 126–132; doi:10.1038/sj.bjp.0701336

RS 57639, by being a partial agonist in rat esophagus but a competitive antagonist in guinea-pig ileum, is one of several ligands which operationally discriminate among 5-HT4 receptors in different tissues. The discovery of splice variants of the 5-HT4 receptor, 5-HT4S and 5-HT4L, raises the possibility that this functional heterogeneity among 5-HT4 receptors may be due to differences in the interaction of ligands with different isoforms of the receptor. To test this idea, the functional and binding interactions of RS 57639 with rat 5-HT4S and 5-HT4L receptors were characterized. RS 57639 stimulated adenylate cyclase in cells expressing 5-HT4S or 5-HT4L receptors with similar potency (pEC50 = 7.9 +/- 0.1 and 7.6 +/- 0.1) and efficacy (71 +/- 3 and 59 +/- 4% of 5-HT). [3H]RS 57639 also bound to 5-HT4S and 5-HT4L receptors with similar affinity (Kd = 0.09 +/- 0.01 and 0.11 +/- 0.01 nM) and specificity (SB204070 > GR113808 > SDZ 205557 > cisapride > renzapride > alpha me-5-HT > 5-CT). Therefore, the operational differences among 5-HT4 receptors, detected with RS 57639, are not explained by differences in the interaction of the ligand with 5-HT4S and 5-HT4L receptors. [3H]RS 57639 binding to guinea-pig striatal membranes was also characterized. [3H]RS 57639 bound with high affinity (Kd = 0.25 +/- 0.07 nM) and a specificity similar to that of the 5-HT4 receptor antagonist, [3H]GR113808. Therefore, while the mechanism by which RS 57639 operationally distinguishes among 5-HT4 receptors was not determined, [3H]RS 57639 was shown to specifically label native and cloned 5-HT4 receptors. As the first selective agonist radioligand to be described for this receptor, [3H]RS 57639 may prove useful in further studies of receptor coupling and ligand interactions.

The 5-HT4 receptor is a member of the seven transmembrane spanning G-protein-coupled family of receptors. The receptor is positively coupled to adenylate cyclase and exists in two isoforms (5-HT4S and 5-HT4L) that differ in the length and sequence of their carboxy termini. The 5-HT4 receptor is pharmacologically defined by selective agonists such as SC 53116 and RS 67506, and selective antagonists such as GR 113808, SB 204070, and RS 39604. The receptor is widely distributed in the central nervous system and peripheral tissues. In the periphery, the receptor plays an important role in the function of several organ responses including the alimentary tract, urinary bladder, heart and adrenal gland. In the alimentary tract, stimulation of 5-HT4 receptors has a pronounced effect on smooth muscle tone, mucosal electrolyte secretion, and the peristaltic reflex. In the urinary bladder, activation of 5-HT4 receptors modulates cholinergic/purinergic transmission. In the heart, stimulation of atrial 5-HT4 receptors produces positive inotropy and tachycardia that can precipitate arrhythmias. In the adrenal gland, agonism of 5-HT4 receptors stimulates release of cortisol, corticosterone, and aldosterone. Since its discovery in 1988, significant advances have been made in our understanding of the physiology and pharmacology of the 5-HT4 receptor. These advances have led to the development of several selective 5-HT4 receptor agonists and antagonists that may have therapeutic utility in the treatment of peripheral disorders such as irritable bowel syndrome, gastroparesis, urinary incontinence and cardiac arrhythmias.

The 5-HT4 receptor is a seven transmembrane spanning, G-protein coupled receptor, that, in all tissues studied to date, positively couples to adenylate cyclase. The receptor exists in two isoforms ...

1. 5-Hydroxytryptamine (5-HT) receptors mediating contraction and relaxation are present in Cynomolgus monkey isolated jugular vein denuded of endothelium. 2. In the absence of spasmogen, alpha-methyl-5-HT and sumatriptan contracted the tissues with potency values (pEC50) of 6.8 (n = 2) and 6.4 +/- 0.1 (mean +/- s.e. mean, n = 3), respectively. In contrast, 5-HT caused an initial contraction (10 nM - 1 microM), followed by relaxation (1 microM - 32 microM). The contractile effect of alpha-methyl-5-HT was antagonized by ketanserin with a pKB value of 8.1 (n = 2). 5-Carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-OH-DPAT did not contract or relax the tissues in the absence of spasmogen. 3. In tissues precontracted with U46619 (10 nM) and in the presence of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, and 5-HT4 receptor blockade, 5-CT and 5-MeOT caused endothelium-independent relaxation with potency values of 7.5 +/- 0.1 (n = 21) and 5.7 +/- 0.1 (n = 4), respectively. The potency of 5-HT was 7.2 (n = 2) while alpha-methyl-5-HT did not start to relax the tissues below a concentration of 10 microM. 4. Relaxations elicited by 5-CT were antagonized by the following compounds (with pKB values in parentheses): methiothepin (9.7), mesulergine (8.1), metergoline (8.0), clozapine (7.8), mianserin (7.7), spiperone (7.3), ritanserin (7.1), methysergide (7.0) and ketanserin (5.7). 5. It is concluded that the 5-HT receptor mediating endothelium-independent relaxation may be a functional correlate of the putative 5-ht7 receptor.

Muscarinic receptors are composed of a family of four subtypes each of which can be distinguished pharmacologically and structurally. The physiological role of each subtype in the central and peripheral nervous systems remains to be clarified, due, in part, to a lack of agonists and antagonists with adequate subtype selectivity. Nonetheless, several agonists with functional selectivity for M1 receptors are now in advanced clinical evaluation for Alzheimer's disease, while selective M1/M3 antagonists may prove useful in the treatment of disorders of smooth muscle function. These novel compounds thus provide an advance over earlier therapeutics with which the clinical efficacy was compromised by the side effect profile. This review attempts to assess novel, selective agonists and antagonists, both in terms of their use in defining muscarinic receptor subtypes and their potential clinical utility.

[3H]GR 113808 binding studies in guinea-pig brain tissue revealed a temperature dependency and pharmacological specificity consistent with labelling of a 5-HT4 receptor. Detailed comparison of competition data between brain regions from human and guinea-pig suggest minor differences in the rank order of affinity. Studies in brain tissue from patients with Alzheimer's, Huntington's and Parkinson's disease have revealed specific deficits in 5-HT4 receptor densities.

The interaction of zamifenacin ((3 R )-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)piperidine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following p K i values; rat cerebral cortex (M 1 ) 7.90 ± 0.08, myocardium (M 2 ) 7.93 ± 0.13, submaxillary gland (M 3 ) 8.52 ± 0.01 and rabbit lung (M 4 ) 7.78 ± 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M 1 ) 7.93 ± 0.09, guinea-pig left atria (M 2 ) 6.60 ± 0.04, guinea-pig ileum (M 3 ) 9.31 ± 0.06, guinea-pig oesophageal muscularis mucosae (M 3 ) 8.84 ± 0.04, guinea-pig trachea (M 3 ) 8.16 ± 0.04, and guinea-pig urinary bladder (M 3 ) 7.57 ± 0.15. Therefore, zamifenacin is selective for muscarinic M 3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M 2 receptors in atria. The degree of muscarinic M 3 M 2 receptor selectivity depends upon the muscarinic M 3 receptor preparation studied.

1. The pharmacology of two novel 5-HT4 receptor agonists, RS 67333 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1(n-butyl)-4-piperidinyl]-1- propanone HCl) and RS 67506 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-(2-methyl sulphonylamino)ethyl-4-piperidinyl]-1-propanone HCl) have been assessed in vitro and in vivo. 2. RS 67333 and RS 67506 exhibited affinities (pKi = 8.7 and 8.8, respectively) for the 5-HT4 binding sites, labelled with [3H]-GR 113808, in guinea-pig striatum. The Hill coefficients from these displacement curves were not significantly different from unity. The compounds exhibited lower affinities (6.0) at several other receptors including 5-HT1A, 5-HT1D, 5-HT2A, 5-HT2C, dopamine D1, D2 and muscarinic M1-M3 receptors. However, RS 67333 and RS 67506 did exhibit affinities for the sigma 1 (pKi = 8.9 and 7.9, respectively) and sigma 2 (pKi = 8.0 and 7.3, respectively) binding sites. 3. At the 5-HT4 receptor mediating relaxation of the carbachol-precontracted oesophagus, RS 67333 and RS 67506 acted as potent (pEC50 8.4 and 8.6, respectively), partial agonists (intrinsic activities, with respect to 5-HT were 0.5 and 0.6, respectively) with respect to 5-HT. Relaxant responses to RS 67333 or RS 67506 were surmountably antagonized by GR 11308 (10 nM), with apparent affinities (pKB) of 9.1 and 9.0, respectively. RS 67333 and RS 67506 induced dose-dependent increases in heart rate of the anaesthetized micropig (ED50 4.9 and 5.4 micrograms kg-1, i.v.), with maximal increases of 35 and 47 beats min-1, respectively. 4. RS 67333 and RS 67506, therefore, acted as potent, partial 5-HT4 receptor agonists in vitro and in vivo. These compounds, by virtue of their high potency and selectivity, may have some utility in elucidating the physiological role of 5-HT4 receptors.

The enantiomers of the indolyl-2-oxoacetamide 5 were found to have 5-HT3 receptor partial agonist activity with (R)-5 having higher potency than (S)-5.

1. Selective antagonism of 5-HT4 receptors may provide therapeutic benefit in certain disorders of the myocardium, alimentary and lower urinary tract. We now report on RS 39604, a novel and selective 5-HT4 receptor antagonist and compare its pharmacological properties with those of SB 204070. 2. In guinea-pig striatal membranes, both RS 39604 and SB 204070 inhibited specific binding of [3H]-GR 113808 in a concentration-dependent manner yielding pKi estimates of 9.1 and 10.9, respectively. RS 39604 displayed a low affinity (pKi < 6.5) for 5-HT1A, 5-HT2C, 5-HT3, alpha 1c, D1, D2, M1, M2, AT1, B1 and opioid mu receptors and moderate affinity for sigma 1, (pKi = 6.8) and sigma 2 (pKi = 7.8) sites. 3. In the rat isolated oesophagus, precontracted with carbachol, RS 39604 (30-300 nM) behaved as a competitive antagonist towards 5-HT-induced relaxation (pA2 = 9.3; Schild slope = 1.0). We and others have shown previously that SB 204070 behaves as an unsurmountable antagonist in this preparation (pA2 approximately 10.5). In the guinea-pig isolated ileal mucosa, RS 39604 (30 nM) antagonized 5-MeOT-induced increase in short-circuit current (pA2 = 9.1). 4. In anaesthetized vagotomized micropigs, RS 39604, administered by the i.v. or intraduodenal (i.duod.) route, produced dose-dependent inhibition of 5-HT-induced tachycardia (ID50 = 4.7 micrograms kg-1, i.v. and 254.5 micrograms kg-1, i.duod). At maximal doses of 30 micrograms kg-1, i.v. and 6 mg kg-1, i.duod., the inhibitory effects of RS 39604 lasted for more than 6 h. In this preparation, SB 204070 was as potent as RS 39604by the i.v. route but was inactive by the intraduodenal route at doses up to 3 mg kg-1.5. In conscious mice, RS 39604, administered by the i.p. or p.o. route, produced dose-depend entinhibition of 5-hydroxytryptophan (5-HTP)-induced diarrhoea (ID50= 81.3 microg kg-1, i.p. and 1.1 mg kg-1,p.o.). In this assay, SB 204070 was inactive by the oral route at doses up to 30 mg kg-1.6. In anaesthetized guinea-pigs, RS 39604 antagonized the contractile effect of 5-HT in the proximal colon by producing parallel, dextral displacement of the dose-response curve to 5-HT. The mean dose ratios to 5-HT at 0.1 mg kg-1, i.v., 1 mg kg-1, i.v. and 10 mg kg-1, i.duod. were 4.6, 30.7 and 10.8,respectively. SB 204070 behaved as an unsurmountable antagonist in this assay.7. In a model of visceral pain in conscious rats, RS 39604 (0.01-1 mg kg-1, i.v.) did not affect colorectal distension-induced increases in arterial pressure whereas morphine (1 mg kg-1, i.v.) produced significant inhibition of the response, implying that 5-HT4 receptors are not involved in nociception in this model.8. The data suggest that RS 39604 is a high affinity and selective 5-HT4 receptor antagonist that is orally active and long-lasting in vivo. It is concluded that RS 39604 may be the preferable probe to use for investigating the physiological and pathophysiological role of 5-HT4 receptors in vivo.

The 5-HT receptor mediating postjunctional relaxation of precontracted guinea-pig ileum has been characterized using several agonists and antagonists. Substance P precontracted tissues were potently relaxed by 5-HT (5-hydroxytryptamine, serotonin), 5-CT (5-carboxamidotryptamine) and several other indoles. The rank order of potency, with pEC50 values in parentheses, was 5-CT (7.6)5-methoxytryptamine (5.7)5-HT (5.5)alpha-methyl-5-HT (4.7)2-methyl-5-HT (4.0) = tryptamine (4.0) = N,N-dimethyl-tryptamine (4.0) = N,N-dimethyl-5-HT (4.0) = dipropyl-5-CT (4.0) = sumatriptan (4.0). 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) acted as a potent (6.3), but partial, agonist with respect to 5-HT. The responses to 5-CT were antagonized by several compounds with the following rank order of affinity, with pKB values in parentheses: LSD (lysergic acid diethylamide; 8.1) = mesulergine (7.8)methysergide (7.6) = spiperone (7.6)clozapine (7.3)(-)-pindolol (6.0)ketanserin (6.0) = ondansetron (6.0) = GR 113808 ([1-(2-methane-sulphonamido-ethyl)-piperidin-4-yl]-methyl-in dole-3- carboxylate maleate;6.0). The relaxant responses to 5-HT were also resistant to tetrodotoxin. These data are consistent with a functional 5-HT receptor, mediating relaxation of guinea-pig ileum, which exhibits an operational profile similar to that of the cloned guinea-pig 5-ht7 receptor. This study, therefore, provides evidence for a functional correlate of the 5-ht7 gene product.

1. In the presence of (-)-cyanopindolol (1.0 microM) and sumatriptan (1.0 microM), 0.5 nM [3H]-carboxamidotrytamine ([3H]-5-CT) labelled a single population of receptors in guinea-pig cerebral cortex membranes. 2. 5-HT-displaceable binding was rapid, saturable and reversible. A high affinity binding site was characterized both by equilibrium saturation (Kd = 0.76 +/- 0.28 nM; Bmax = 68.1 +/- 26.7 fmol mg-1 protein) and kinetic (Kd = 0.18 +/- 0.05 nM) analysis. The pharmacological profile of this site was similar to the profile obtained in transfected CHO-K1 cells expressing guinea-pig 5-ht7 receptors. 3. Autoradiographic analysis revealed a discrete localization of binding sites in guinea-pig brain, with the highest density of sites in the medial thalamic nuclei and related limbic and cortical regions. Moderate levels of binding were detected in sensory relay nuclei, substantia nigra, hypothalamus, central grey and dorsal raphe nuclei. This distribution corresponded to that observed using in situ hybridization with [35S]-UTP labelled riboprobes complementary to mRNA encoding the guinea-pig 5-ht7 receptor. 4. In conclusion, under appropriate conditions, [3H]-5-CT labelled a single population of saturable binding sites that corresponded to an endogenous 5-ht7 receptor in guinea-pig brain. The distribution of 5-ht7 receptors in thalamocortical and limbic brain regions suggests a role for these receptors in sensory and affective behaviours.

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M1 ,M 2 ,M 3 ,M 4, and M5 muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques. Curr. Protoc. Pharmacol. 48:4.15.1-4.15.29. C � 2010 by John Wiley & Sons, Inc.

Structural modification of the 2-methoxy group and at the 4-position of the piperidine ring of the 5-HT4 partial agonist 1 led to analogues with increased affinity for the 5-HT4 receptor and loss of agonist activity. Similar modification of 2 resulted in 2-(3,5-dimethoxy) benzyloxy derivatives (23, 24, 26–28) that were found to be 5-HT4 receptor antagonists with subnanomolar affinity.

The ketone analogue 5 of the 5-HT4 receptor antagonist RS-23597 (1) was found to be a 5HT4 receptor partial agonist. Structure-activity relationships developed for compound 5 led to the synthesis of 4-piperidinyl derivatives (15–18, 20) that were found to be partial agonists with increased affinity.

Recent radioligand binding studies have demonstrated the presence of 5-HT 4 receptors throughout the nigrostriatal and mesolimbic systems of mammalian brain. In many regions, the binding has not yet been correlated with functional responses. The present study was carried out to fully characterize the regional distribution of 5-HT 4 receptors in brain sections from three species using two structurally distinct radioligands, [ 3 H]GR113808, and [ 3 H]BIMU-1. The highest density of 5-HT 4 receptors labeled with [ 3 H]GR113808 was found in the olfactory tubercle, substantia nigra, ventral pallium and striatum of rat and guinea pig, and similar regions of pig-tail macaque monkey. A similar distribution of 5-HT 4 receptors was observed in guinea pig brain using [ 3 H]BIMU-1. With either ligand, the binding was saturable and of high affinity ( K d = 0.08–0.53 nM for [ 3 H]GR113808; 1.4–3.0 nM for [ 3 H]BIMU-1). These results extend previous distribution studies, confirm the heterogenous distribution of 5-HT 4 receptors throughout the nigrostriatal and mesolimbic systems of three species, and demonstrate a similar distribution using two structurally distinct 5-HT 4 radioligands.

The pharmacological properties of the (R) and (S) enantiomers of RS 56532 have been studied in vitro and in vivo. In radioligand binding studies at 5-HT4 receptors in guinea-pig striatum, (S) RS 56532 exhibited a higher affinity than (R) RS 56532 (-log Ki = 7.6 and 6.5, respectively). (S) RS 56532 acted as a potent agonist at 5-HT4 receptors mediating relaxation of rat oesophageal muscularis mucosae (-log EC50 = 7.9) while (R) RS 56532 acted as a weaker agonist at this receptor (-log EC506.0). These data suggest that at 5-HT4 receptors, the enantiomeric selectivity of RS 56532 was (S)(R). In binding studies at 5-HT3 receptors in rat cortex, (R) RS 56532, conversely, exhibited a higher affinity than (R) RS 56532 (-log Ki = 9.1 and 8.0, respectively). At 5-HT3 receptors in guinea-pig isolated ileum, (R) RS 56532 exhibited an affinity (-log KB) of 7.9, whereas (S) RS 56532 (1 nM-1 microM) was inactive. No agonism was observed at ileal 5-HT3 receptors with either enantiomers. These data suggest that at 5-HT3 receptors in rat and guinea-pig, both enantiomers acted as antagonists, with (R)(S) RS 56532. At the non-5-HT3, high affinity '(R) zacopride' site, (R) RS 56532 exhibited a higher affinity than (S) RS 56532 (-log Ki = 6.1 and 4.9). This site was insensitive to potent 5-HT3 antagonists such as (R) YM 060 or ondansetron. However, it was recognized with relatively high affinity (-log Ki = 7.5) by the (R), but not (S) enantiomer, of RS 42358 (-log Ki = 4.7). Since (S) RS 42358 is a high affinity 5-HT3 receptor antagonist, these data further highlight the dissimilarity between the 5-HT3 receptor and the '(R) zacopride' site. The '(R) zacopride' site also appeared to be pharmacologically distinct from the 5-HT4 receptor, since 5-HT4 ligands such as renzapride, SDZ 205,557 or RS 23597-190 exhibited low affinities. The enantiomeric selectivity of (R) and (S) RS 56532 in vivo was consistent with findings in vitro. At 5-HT4 receptors mediating tachycardia in the pig, 5-HT induced a dose-dependent tachycardia (ED50 = 3 micrograms kg-1, i.v.; maximum response = 90-100 beats min-1). (S) RS 56532 increased heart rate by 88 min-1 with a potency of (ED50) of 3 micrograms kg-1, i.v.(ABSTRACT TRUNCATED AT 400 WORDS)

The physiological role of muscarinic receptors is highly complex and, although not completely understood, has become clearer over the last decade. Recent pharmacological evidence with novel compounds, together with data from transgenic mice, suggests that all five subtypes have defined functions in the nervous system as well as mediating the non neuronal, hormonal actions of acetylcholine. Numerous novel agonists, allosteric regulators, and antagonists have now been identified with authentic subtype specificity in vitro and in vivo. These compounds provide additional pharmacological opportunities for selective subtype modulation as well as a new generation of muscarinic receptor-based therapeutics.

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 KD= 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.

The binding of three, structurally distinct, 5-hydroxytryptamine, (5-HT3) receptor radioligands was characterized in rat cerebral cortex, rabbit ileum myenteric plexus and NG-108-15 neuroblastoma cells. The density of sites labeled by the three ligands in rat cortex or in rabbit ileum was markedly different. [3H]Quipazine labeled more sites than [3H]GR 65630 in rat cortex (4.0-fold) and rabbit ileum (1.8-fold), but not in NG-108-15 cells. [3H]Quipazine also labeled a greater density of sites than [3H]gr3nisectron in rat cortex (7-fold) but not in NG-108 cells. [3H]Quipazine binding in rat cortex and rabbit ileum, but not in NG-108-15 cells, was displaced by non-radiolabeled GR 65630 in a manner consistent with an interaction with more than one site. These data indicate that not all 5-HT3 receptor radioligands recognize the same population of 5-HT3 binding sites with equivalent density and further suggest the existence of subtypes of 5-HT3 receptor binding sites in rat cortical and rabbit myenteric plexus preparations.

Several series of N-(quinuclidin-3-yl)aryl and heteroaryl-fused pyridones were synthesized and evaluated for 5-HT3 receptor affinity. In the heteroaryl series, 2-(quinuclidin-3-yl)tetrahydropyrido-[4,3-b]indol-1-one (8a) and the 4,5-alkano-bridged analogues (14 and 15) displayed high 5-HT3 receptor affinity with pKi values > 9. The (3S)-quinuclidinyl isomers had > 10 fold higher affinity than the (3R)-isomers. In a series of 2-quinuclidin-3-yl)isoquinolin-1-ones, derivatives substituted with small lipophilic groups (25b-e) and with 4,5-alkano-bridges (34-36) also displayed high affinity. In particular, the hexahydro-1H-benz[de]isoquinolinone (S,S)-37 was the highest affinity 5-HT3 receptor ligand prepared (pKi 10.4). A number of the high affinity ligands were shown to be potent 5-HT3 receptor antagonists in vivo as determined by inhibition of the B-J reflex in the anesthetized rat. Again, (S,S)-37 was the most active agent tested (ID50 0.02 microgram/kg i.v.), and this compound was also potent in blocking cisplatin-induced emesis in both the ferret and the dog. Computer modeling studies were performed, and previously reported 5-HT3 receptor antagonist pharmacophore models were refined to include a key lipophilic binding domain.

The objective of this study was to explore the role of 5-HT3 receptors in modulating potassium (K+)-evoked release of [3H]-acetylcholine ([3H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [3H]-choline. Rat entorhinal cortices were cross-chopped into 300 microns slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/l Ca2+ and stimulated with two consecutive exposures of 20 mmol/l K+ for 4 min (S1 and S2, respectively). Compounds were added 20 min before S2 stimulation and remained in the superfusion buffer for the duration of the experiment. The S2/S1 ratio was then calculated. Stimulated release of [3H]-ACh was dependent on extracellular Ca2+ and K+ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10(-9)-10(-6) mol/l), in the presence of 1 mumol/l ritanserin or 1 mumol/l ondansetron, had no influence on K(+)-evoked release of [3H]-ACh. In slices prepared from Hooded Lister rats, 2 mumol/l 5-HT but not 2-Me-5-HT significantly (P0.05) inhibited K(+)-evoked [3H]-ACh release only 17% in the presence of 1 mumol/l ritanserin. However, 2 mumol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K(+)-evoked ACh release was observed with either 5-HT3 receptor agonists or antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

RS-42358-197[(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1 H-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 (pKi) of 9.8 +/- 0.1, while having weak affinity (pKi6.0) 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 (KD) of 0.12 +/- 0.01, 0.20 +/- 0.01, and 0.10 +/- 0.01 nM and densities (Bmax) 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)-zacopridetropisetron(R)-zacoprideondansetronMDL722225-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 (Bmax = 91 +/- 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (KD = 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.

The S-isomer of the novel 5-HT3 receptor antagonist RS-42358 ((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1-H- benzo[de]isoquinolin-1-one, RS-42358-197) disinhibited behaviour in the mouse suppressed by the aversive situation of the light/dark test box. RS-42358-197 was effective at sub-ng/kg dose levels and the efficacy was maintained over a 100 million-fold dose range. In contrast, the R-isomer was ineffective at all doses studied. The S-isomer also disinhibited a suppressed behaviour in social interaction and elevated X-maze tests in the rat and reduced anxiety-related behaviours in a marmoset human threat test. RS-42358-197 prevented the exacerbation of the suppression of behaviour in the mouse light/dark test following withdrawal from treatment with alcohol, nicotine, cocaine and diazepam. Thus, the S-isomer of RS-42358 has a consistent non-sedating anxiolytic profile in rodent and primate models. It is exceptionally potent and a maintained efficacy at high doses distinguishes its actions from many other 5-HT3 receptor antagonists.

1. The interaction of the novel antagonist, SDZ 205,557 (2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester), at 5-HT3 and 5-HT4 receptors has been assessed in vitro and in vivo. 2. In guinea-pig hippocampus and in the presence of 0.4 microM 5-carboxamidotryptamine, 5-HT4-mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol-contracted oesophagus, 5-HT4-receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist-independent with the exception of (R)-zacopride, against which a significantly lower value (6.4) was observed. 3. In functional studies of 5-HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea-pig ileum compared with 6.9 at binding sites labelled by [3H]-quipazine in NG108-15 cells. In the anaesthetized, vagotomized micropig, SDZ 205,557 produced only a transient blockade of 5-HT4-mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half-lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively. 4. In conclusion, SDZ 205,557 has similar affinity for 5-HT3 and 5-HT4 receptors. The apparent selectivity observed in guinea-pig is due to the atypical nature of the 5-HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5-HT4 receptor.

The enantiomers of the indolyl-2-oxoacetamide 5 were found to have 5-HT3 receptor partial agonist activity with (R)-5 having higher potency than (S)-5.

1. The effects of acute desensitization of muscarinic receptors mediating contractile responses of the guinea-pig ileal longitudinal muscle were studied in vitro, using similar conditions for both functional and radioligand binding studies. 2. The pA2 values for a number of muscarinic antagonists (pirenzepine, methoctramine, (+/-)para-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methyl piperidine-methiodide) indicated that the contractile response to carbachol was mediated through an M3 muscarinic receptor. In binding experiments the muscarinic receptor subtype population in ileal longitudinal muscle was found to be heterogeneous, consisting of approximately 77% M2 and 23% M3 receptors. 3. Pre-exposure of ileal longitudinal muscle to 10 microM carbachol for 30 min produced a reduction (28 +/- % of control maximum) in the maximum contractile response and a dextral shift in the concentration-effect curve to carbachol. Prior equilibration (60 min) with (+/-)p-F-HHSiD (1 microM), but not with methoctramine (1 microM) or pirenzepine (0.3 microM), prevented the desensitization. Desensitization under these conditions did not alter either the apparent affinity, the total number of binding sites or the relative, proportions of M2 and M3 muscarinic receptors, as determined in radioligand binding studies. Desensitization did not cause any meaningful change in either the apparent affinity of carbachol or the proportion of the high and low affinity binding sites. 4. It is concluded that desensitization of the contractile responses of the guinea-pig ileal longitudinal muscle is a result of M3 but not M2 muscarinic receptor desensitization. Acute desensitization, therefore, is not accompanied by meaningful changes in the total number of both M2 and M3 receptors or by alterations in the affinity of the receptor to ligands.

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner. Binding to this site was potently inhibited by 5-HT uptake blockers such as paroxetine and fluoxetine (pKi s = 8.6-9.9), while 5-HT3 receptor ligands exhibited only low affinity (pK;7). No detectable specific [3H]-paroxetine binding was observed in NG108-15 or NCB-20 cell homogenates. 4. [3H]-quipazine binding to homogenates of NG108-15, NCB-20 cells and rat cortex (in the presence of 0.1 microM paroxetine) exhibited similar pharmacological characteristics. 5-HT3 receptor antagonists competed for [3H]-quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: (S)-zacopridequarternized ICS 205-930 2 granisetronondansetronICS 205-209 (R)-zacopridequipazinerenzaprideMDL-72222butanopridemetoclopramide. 5. [3H]-GR65630 labelled a site in NCB-20 cell homogenates with an affinity (Kd = 0.7 + 0.1 nms n = 4) and density (B__ = 1800 + 1000 fmol mg- protein) comparable to that observed with [3H]-quipazine. Competition studies also indicated a good correlation between the pharmacology of 5-HT3 binding sites when [3H]-GR65630 and [3H]-quipazine were used in these cells. 6. In conclusion, [3H]-quipazine labelled 5-HT3 receptor sites in homogenates of NG108-15 cells, NCB-20 cells and rat cerebral cortex. In rat cortical homogenates, [3H]-quipazine also bound to 5-HT uptake sites, which could be blocked by 0.1 microM paroxetine. The pharmacological specificity of the 5-HT3 receptor labelled by [3H]-quipazine was similar in the neuroblastoma cells and rat cortex and was substantiated in NCB-20 cells by the binding profile of the selective 5-HT3 receptor antagonist, [3H]-GR65630.