An outbreak of SARS-CoV-2 in a public-facing office in England.

BACKGROUND: An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an attack rate of 55% (22/40 workers) occurred at a public-facing office in England from August to September 2021. Published evidence regarding outbreaks in office workplaces remains limited. AIMS: To describe an investigation of workplace- and worker-related risk factors following an outbreak of SARS-CoV-2 in a public-facing office. METHODS: The COVID-19 (coronavirus disease 2019) Outbreak Investigation to Understand Transmission (COVID-OUT) study undertook an investigation of the outbreak. This included surface sampling, occupational environmental assessment, molecular and serological testing of workers, and detailed questionnaires. RESULTS: Despite existing COVID-19 control measures, surface sampling conducted during a self-imposed 2-week temporary office closure identified viral contamination (10/60 samples, 17% positive), particularly in a small, shared security office (6/9, 67% positive) and on a window handle in one open-plan office. Targeted enhanced cleaning was, therefore, undertaken before the office reopened. Repeat surface sampling after this identified only one positive (2%) sample. Ventilation was deemed adequate using carbon dioxide monitoring (typically ≤1000 ppm). Twelve workers (30%) responded to the COVID-OUT questionnaire, and all had been vaccinated with two doses. One-third of respondents (4/12) reported direct physical or close contact with members of the public; of these, 75% (3/4) reported a divider/screen between themselves and members of the public. CONCLUSIONS: The results highlight the potential utility of surface sampling to identify SARS-CoV-2 control deficiencies and the importance of evolving, site-specific risk assessments with layered COVID-19 mitigation strategies.

A follow up study of occupational exposure to 4,4'-methylene-bis(2-chloroaniline) (MbOCA) and isocyanates in polyurethane manufacture in the UK.

This is a follow up survey of exposure to 4,4'-methylene-bis(2-chloroaniline) (MbOCA) and isocyanates in the UK polyurethane industry. Urine samples (n=446) were collected from 90 different workers. MbOCA levels were below the limit of detection in 170 samples and 26 were above the UK Biological Monitoring Guidance Value (BMGV) of 15 µmol MbOCA/mol creatinine. Detailed advice and guidance was given to each workplace at the end of the survey in 2008 and the 90% value reduced from 10 to 3 µmol MbOCA/mol creatinine in samples collected since. There was a positive correlation between glove contamination and urinary MbOCA and levels were dependent upon individual working practices especially how gloves were used. Of the 446 samples analysed for urinary metabolites of toluene diisocyanate 280 were below the detection limit and 126 were above the BMGV (1 µmol/mol creatinine). Of the 326 urine samples that were analysed for metabolites of methylenediphenyl diisocyanate, 270 were below the detection limit and 13 were above the BMGV for isocyanates. There was no correlation between urinary levels of isocyanates and MbOCA suggesting different routes of absorption, most likely inhalation and dermal respectively.

A 100K well screen for a muscarinic receptor using the Epic label-free system--a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors.

Seven-transmembrane receptors (7TMRs) are a family of proteins of great interest as therapeutic targets because of their abundance on the cell surface, diverse effects in modulating cell behavior and success as a key class of drugs. We have evaluated the Epic label-free system for the purpose of identifying antagonists of the muscarinic M3 receptor. We compared the data generated from the label-free technology with data for the same compounds in a calcium flux assay. We have shown that this technology can be used for high throughput screening (HTS) of 7TMRs and as an orthogonal approach to enable rapid evaluation of HTS outputs. A number of compounds have been identified which were not found in a functional HTS measuring the output from a single pathway, which may offer new approaches to inhibiting responses through this receptor.

Regulation of BAG-1 IRES-mediated translation following chemotoxic stress.

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5'-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES-meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.

Initiation factor modifications in the preapoptotic phase.

Recent studies have identified several mechanistic links between the regulation of translation and the process of apoptosis. Rates of protein synthesis are controlled by a wide range of agents that induce cell death, and in many instances, the changes that occur to the translational machinery precede overt apoptosis and loss of cell viability. The two principal ways in which factors required for translational activity are modified prior to and during apoptosis involve (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. In this review, we summarise the principal targets for such regulation, with particular emphasis on polypeptide chain initiation factors eIF2 and eIF4G and the eIF4E-binding proteins. We indicate how the functions of these factors and of other proteins with which they interact may be altered as a result of activation of apoptosis and we discuss the potential significance of such changes for translational control and cell growth regulation.

The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock.

BAG-1 (also known as RAP46/HAP46) was originally identified as a 46 kDa protein that bound to and enhanced the anti-apoptotic properties of Bcl-2. BAG-1 exists as three major isoforms (designated p50, p46 and p36 or BAG-1L, BAG-1M and BAG-1S respectively) and one minor isoform (p29), which are translated from a common transcript. The differing amino terminus determines both the intracellular location and the repertoire of binding partners of the isoforms which play different roles in a variety of cellular processes including signal transduction, heat shock, apoptosis and transcription. Although in vitro data suggest that the four BAG-1 isoforms are translated by leaky scanning, the patterns of isoform expression in vivo, especially in transformed cells, do not support this hypothesis. We have performed in vivo analysis of the BAG-1 5' untranslated region and shown that translation initiation of the most highly expressed isoform (p36/BAG-1S) can occur by both internal ribosome entry and cap-dependent scanning. Following heat shock, when there is a downregulation of cap-dependent translation, the expression of the p36 isoform of BAG-1 is maintained by internal ribosome entry.

1,3-Biarylureas as selective non-peptide antagonists of the orexin-1 receptor.

This communication reports SARs for the first orexin-1 receptor antagonist series of 1-aryl-3-quinolin-4-yl and 1-aryl-3-naphthyridin-4-yl ureas. One of these compounds, 31 (SB-334867), has excellent selectivity for the orexin-1 receptor, blood-brain barrier permeability and shows in vivo activity following ip dosing.

Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras.

It has been reported previously that the 5' untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Comparative pharmacological study of ropinirole (SKF-101468) and its metabolites in rats.

The dopamine receptor agonist ropinirole (SKF-101468) is used to treat Parkinson's disease. Ropinirole is metabolized by two routes to a series of different metabolites although the predominant pathway is species-dependent. It is unknown whether any of the metabolites contribute to its antiparkinsonian activity and whether D3 or D2 receptor agonist activity plays a preferential role. Therefore ropinirole and its primary metabolites, SKF-104557, SKF-97930 and SKF-96990, and the rat metabolite, SKF-89124 were tested in the 6-hydroxydopamine lesion model of Parkinson's disease. SKF-89124 and SKF-96990 were also assayed in radioligand binding and microphysiometer functional assays at cloned human dopamine D2 and D3. Ropinirole and SKF-89124 were equipotent in-vivo, and produced dose-related increases in circling at 0.05-0.8 mg kg(-1), s.c. (ropinirole) and 0.05-0.75 mg kg(-1), s.c. (SKF-89124). Neither SKF-96990 or SKF-97930, at doses up to 15 mg kg(-1), increased the circling rate. Some circling was observed with 15 mg kg(-1) SKF-104557 but the response was less than half that produced by ropinirole (0.8 mgkg(-1)). SKF-104557 was 150-fold less potent than ropinirole. SKF-89124 possessed-30-fold higher affinity for D3 over D2 receptors in radioligand binding studies, but was not selective in the functional microphysiometer assay. SKF-96990 was 10-fold selective for D3 over D2 receptors in the radioligand binding assay. Ropinirole and SKF-104557 are 20-fold selective for D3 over D2 receptors in radioligand binding assays whereas in microphysiometry, selectivity is 10-fold. SKF-97930 is inactive in radioligand binding and microphysiometer assays. Primary metabolites of ropinirole did not contribute significantly to its activity in this model of Parkinson's disease. The lack of dopamine D3/D2 receptor selectivity for ropinirole rules out the possibility of attributing the degree of either D2 or D3 receptor activity to the behavioural efficacy of ropinirole.

Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A.

SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Initiation of Apaf-1 translation by internal ribosome entry.

The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5' untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5' UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3' end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised. Oncogene (2000) 19, 899 - 905.

Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment.

The 5' UTR of c -myc mRNA contains an internal ribo-some entry segment (IRES) and consequently, c -myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c -myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c- myc 5' UTR, we demonstrate that both mechanisms can contribute to c- myc protein synthesis. A wide range of cell types are capable of initiating translation of c- myc by internal ribosome entry, albeit with different efficiencies. Moreover, our data suggest that the spectrum of efficiencies observed in these cell types is likely to be due to variation in the cellular concentration of non-canonical translation factors. Interestingly, the c -myc IRES is 7-fold more active than the human rhinovirus 2 (HRV2) IRES and 5-fold more active than the encephalomyocarditis virus (EMCV) IRES. However, the protein requirements for the c -myc IRES must differ significantly from these viral IRESs, since an unidentified nuclear event appears to be a pre-requisite for efficient c -myc IRES-driven initiation.

Site-specific splice variation of the human P2X4 receptor.

P2X4 receptors are expressed in specific brain areas. We now describe site-specific splice variations of the human P2X4 receptor subunit, occurring at residue [YVIG / WVFV(W)] near the end of the first predicted transmembrane domain. p2X4(b) is formed by the insertion of an additional 16 amino acids. p2X4(C) is formed by deleting a cassette of 130 amino acids, including six of the 10 conserved extracellular cysteine residues. Transfection of P2X4(a), but not p2x4(c), formed functional channels in Xenopus oocytes and human 1321N1 cells. After transfection of p2X4(b) small, inconsistent ATP-evoked responses were detected only in the human cells, but when co-expressed, p2x4(b) may alter the function of P2X4(a) in oocytes. The distribution of splice variant RNA within human brain suggests regionally-dependent expression. These data indicate that the functions of the human P2X4 receptor may be altered by alternative splicing.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (-)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

1. The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF-104557 (4-[2-(propylamino)ethyl]-2-(3H) indolone), SKF-97930 (4-carboxy-2-(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry. 2. All the receptor agonists tested (ropinirole, SKF-104557, SKF-97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members. 3. At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF-104557, were partial agonists at the hD4 receptor. 4. Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s. 5. The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities. 6. The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF-104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound.

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D3 receptor.

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF3SO2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D2 receptor.

Gamma-aminobutyric acidA receptor function is desensitised in rat cultured cerebellar granule cells following chronic flunitrazepam treatment.

This study examined gamma-aminobutyric acidA (GABA(A)) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the alpha1 and beta2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced (p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate (Emax) (16.5 +/- 1.2% and 11.3 +/- 1.0%, 2-day control and treated cells, respectively; 17.4 +/- 1.0% and 9.9 +/- 0.7%, 7-day control and treated cells, respectively; best-fit Emax +/- SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 +/- 1.7 and 2.3 +/- 1.8 microM, 2-day control and treated cells, respectively; 1.7 +/- 1.5 and 1.5 +/- 1.5 microM, 7-day control and treated cells, respectively; best-fit EC50 +/- SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA(A) receptor alpha1 and beta2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA(A) receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA(A) receptor function.

Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors. Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes. Carbachol and xanomeline stimulated basal [35S]-GTPgammaS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPgammaS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 microM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors. In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5-HT2A, h5-HT2B and h5-HT2c receptors with potencies similar to its affinity at these receptors. These studies indicate that xanomeline is a potent agonist at 5-HT1A and 5-HT1B receptors and an antagonist at 5-HT2 receptor subtypes.