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Radioligand Binding (radioligand + binding)

Distribution by Scientific Domains
Medical Sciences68%
Chemistry18%
Life Sciences13%

Terms modified by Radioligand Binding

  • radioligand binding studies
    		•

    Selected Abstracts

    Novel estrogen receptor ligands and their structure,activity relationship evaluated by scintillation proximity assay for high-throughput screening

    DRUG DEVELOPMENT RESEARCH, Issue 4 2005
    Ling He
    Abstract The estrogen receptor (ER) is an important drug target with allosteric characteristics that binds orthotopic hormones and other ligands. A recently developed scintillation proximity (SPA)-based assay for high-throughput screening (HTS) of compound libraries was used to identify novel estrogen receptor ligands that might have ER subtype selective binding activity. Radioligand binding was determined in a multi-detector scintillation counter designed for microtitration plates. Equilibrium binding experiments and kinetic competition tests were performed with [3H]-estradiol and human ER, and ER, receptors. A library of 6,000 structurally diverse compounds was screened. From this, several novel ligands were identified that showed pronounced subtype-selective differences in ligand binding for ER, and ER,. The observed equilibrium dissociation constant (Kd) for the binding of [3H]estradiol to ER, and ER, receptors were approximately 0.25 and 0.64 nM, respectively. When 17,-estradiol, raloxifene and daidzein were tested for binding affinity to ER, in a competition assay, the IC50 values were 0.34, 1.31, and 75.6 nM, respectively. When tested for binding affinity to ER,, the IC50 values were 1.05, 11.4, and 10.6 nM, respectively. The results obtained show that the methodology is valid in comparison to previously published data regarding estradiol and other standard compounds (raloxifene and daidzein) binding characteristics of estrogen receptors. The assay is also well suited to applied research as a tool in HTS of compound libraries in the search of ER ligands. Several novel active compounds were identified and selected as potent ER subtype ligands. Drug Dev Res 64:203,212, 2005. © 2005 Wiley-Liss, Inc. [source]

    MDMA self-administration in rats: acquisition, progressive ratio responding and serotonin transporter binding

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007
    Susan Schenk
    Abstract 3,4-Methylenedioxymethamphetamine (MDMA) self-administration has been shown in animals with extensive drug histories, but only a small number of studies have examined high rates of responding maintained by MDMA in previously drug-naïve animals. In the present study, influence of dose (0.25 or 1.0 mg/kg/infusion) on the acquisition of MDMA self-administration was measured during daily 6-h sessions. Dose,effect data were obtained for MDMA (0.25,1.0 mg/kg/infusion) self-administration under a progressive ratio (PR) schedule of reinforcement. The effect of experimenter- or self-administered MDMA on [3H] paroxetine binding in several brain regions was measured. Acquisition of MDMA self-administration was highly variable and not different for 0.25 or 1.0 mg/kg/infusion progressed with approximately 60% of the rats acquiring reliable self-administration during the 15-day test period. The percentage of rats that acquired MDMA self-administration was lower than the percentage of rats that acquired cocaine (0.5 mg/kg/infusion) self-administration, and cocaine self-administration was acquired with a shorter latency. Responding maintained by MDMA was dose dependent, and breakpoints under a PR schedule increased with dose. Radioligand binding and autoradiography demonstrated lower densities of serotonin transporter sites (SERT) in MDMA self-administering rats as compared with controls across brain regions. The reduction in SERT densities was comparable in magnitude to rats treated with experimenter-administered doses of MDMA. These data support the idea that MDMA is a drug with high abuse liability, and long-term self-administration may lead to long-lasting deficits in serotonin neurotransmission. [source]

    Acute sleep-promoting action of the melatonin agonist, ramelteon, in the rat

    JOURNAL OF PINEAL RESEARCH, Issue 2 2008
    Simon P. Fisher
    Abstract:, Insomnia, which is severe enough to warrant treatment, occurs in ,10% of the general population. It is associated with a range of adverse consequences for human health, economic productivity and quality of life. In animal and human studies, administration of melatonin has been reported to promote sleep, although there has been controversy regarding its effectiveness. The present study used a chronically implanted radiotelemetry transmitter to record electroencephalogram (EEG) and electromyogram (EMG) to enable discrimination of wake (W), nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in un-restrained rats. The acute action of melatonin and ramelteon, a melatonin agonist recently approved for long-term treatment of insomnia in the USA, was examined. Radioligand binding assays on recombinant human MT1/MT2 receptors showed that both the melatonin and ramelteon were both high affinity, nonsubtype selective ligands. Both compounds acted as potent full agonists on a cellular model of melatonin action, the pigment aggregation response in Xenopus laevis melanophores. Both melatonin and ramelteon (10 mg/kg, i/p), administered close to the mid-point of the dark phase of the L:D cycle, significantly reduced NREM sleep latency (time from injection to the appearance of NREM sleep). Both the drugs also produced a short-lasting increase in NREM sleep duration, but the NREM power spectrum was unaltered. Neither drug altered REM latency, REM sleep duration nor power spectrum during REM sleep. In conclusion, ramelteon administration, like melatonin, exerted an acute, short-lasting sleep-promoting effect in the rat, the model most commonly used to evaluate the activity of novel hypnotic drugs. [source]

    M2 mediated contractions of human bladder from organ donors is associated with an increase in urothelial muscarinic receptors,

    NEUROUROLOGY AND URODYNAMICS, Issue 1 2007
    Alan S. Braverman
    Abstract Aims Previous studies have shown increased density of M2 receptors in hypertrophied rat bladders that possess an M2 contractile phenotype. The aim of the current study is to determine whether human bladders with an M2 contractile phenotype also have a greater density of bladder M2 receptors. Materials and Methods Human bladders were obtained from 24 different organ transplant donors. Darifenacin and methoctramine affinity was determined by the rightward shift of cumulative carbachol concentration contractile response curves for each bladder. Radioligand binding and immunoprecipitation was used to quantify M2 and M3 subtypes in isolated detrusor muscle and urothelium. In addition, pig bladder muscle and urothelial receptors were quantified for comparison. Results In the human urothelium total, M2 and M3 muscarinic receptor density is significantly negatively correlated with the affinity of darifenacin for inhibition of contraction of the detrusor muscle. In the detrusor muscle there is no correlation between receptor density and darifenacin affinity for inhibition of contraction. Muscarinic receptor density is greater in the muscle than in the urothelium in human bladders whereas in the pig bladder the density is greater in the urothelium than in the muscle. Conclusions The greater density of urothelial muscarinic receptors in human bladders with lower darifenacin affinity, indicative of a greater contribution of M2 receptors to the contractile response, points towards a possible role of the urothelium in controlling M2 mediated contractile phenotype. In comparison between human and pig bladders, the distribution of muscarinic receptor subtypes in the muscle and urothelium are quite different. Neurourol. Urodynam. © 2006 Wiley-Liss, Inc. [source]

    Pharmacological and Functional Characterization of Novel EP and DP Receptor Agonists: DP1 Receptor Mediates Penile Erection in Multiple Species

    THE JOURNAL OF SEXUAL MEDICINE, Issue 2 2008
    Nadia Brugger PhD
    ABSTRACT Introduction., Despite the widespread use of prostaglandin E1 as an efficacious treatment for male erectile dysfunction for more than two decades, research on prostanoid function in penile physiology has been limited. Aim., To characterize the pharmacological and physiological activity of novel subtype-selective EP and DP receptor agonists. Methods., Radioligand binding and second messenger assays were used to define receptor subtype specificity of the EP and DP agonists. Functional activity was further characterized using isolated human and rabbit penile cavernosal tissue in organ baths. In vivo activity was assessed in rabbits and rats by measuring changes in cavernous pressure after intracavernosal injection of receptor agonists. Main Outcome Measures., Receptor binding and signal transduction, smooth muscle contractile activity, erectile function. Results., In organ bath preparations of human cavernosal tissue contracted with phenylephrine, EP2- and EP4-selective agonists exhibited variable potency in causing relaxation. One of the compounds caused mild contraction, and none of the compounds was as effective as PGE1 (EC50 = 0.23 µM). There was no consistent correlation between the pharmacological profile (receptor binding and second messenger assays) of the EP agonists and their effect on cavernosal tissue tone. In contrast, the DP1-selective agonist AS702224 (EC50 =29 nM) was more effective in relaxing human cavernosal tissue than either PGE1, PGD2 (EC50 = 58 nM), or the DP agonist BW245C (EC50 =59 nM). In rabbit cavernosal tissue, PGE1 and PGD2 caused only contraction, while AS702224 and BW245C caused relaxation. Intracavernosal administration of AS702224 and BW245C also caused penile tumescence in rabbits and rats. For each compound, the erectile response improved with increasing dose and was significantly higher than vehicle alone. Conclusions., These data suggest that AS702224 is a potent DP1-selective agonist that causes penile erection. The DP1 receptor mediates relaxation in human cavernosal tissue, and stimulates pro-erectile responses in rat and rabbit. Thus, rabbits and rats can be useful models for investigating the physiological function of DP1 receptors. Brugger N, Kim NN, Araldi GL, Traish AM, and Palmer SS. Pharmacological and functional characterization of novel EP and DP receptor agonists: DP1 receptor mediates penile erection in multiple species. J Sex Med 2008;5:344,356. [source]

    Structure,activity relationships of isoeugenol-based chlorophenylpiperazine derivatives on serotonergic/adrenergic receptor, platelet aggregation, and lipid peroxidation

    DRUG DEVELOPMENT RESEARCH, Issue 5 2010
    Kuo-Ping Shen
    Abstract Three isoeugenol-based eugenosedin chlorphenylpiperazine derivatives, Eu-A, Eu-B, and Eu-C, were synthesized and tested for their serotonergic, adrenergic antagonist, antioxidant, and anti-aggregation activities. In radioligand binding assays, all three agents displayed significant binding affinities on ,1, ,2, ,1, 5-HT1B, and 5-HT2A receptors. In human platelet, they inhibited epinephrine and 5-HT-induced aggregation, and in human platelet with ,2 and 5-HT2A receptors they had a competitive binding effect. Eu-B and Eu-C were more potent than Eu-A. All compounds had antioxidant effects derived from aryloxypropanolamine. Eu- A, Eu-B, or Eu-C (1, 3, 5,mg/kg iv) given to normotensive Wistar rats produced a dose-dependent decrease in mean arterial blood pressure and heart rate and when injected into the cisternum, Eu-A, Eu-B, or Eu-C (0.3, 0.03,µmol) increased blood pressure within 15,min. Pretreatment with any of the three agents inhibited clonidine (38,pmol)-induced hypotension. In vitro experiments, Eu-A, Eu-B, or Eu-C (1, 10, and 100,µM) competitively antagonized norepinephrine-, clonidine-, and 5-HT (10,8,10,4,M)-induced vasocontraction in isolated rat aorta, and competitively antagonized isoproterenol (10,8,10,4,M)-induced positive inotropic effects in a concentration-dependent manner in the isolated rat left atrium. In isolated rabbit ear arteries sensitized with 16,mM K+, all three agents antagonized 5-nonyloxytryptamine- and 5-HT-induced vasocontractions. These findings show that Eu-A, Eu-B, and Eu-C possess functional ,1, ,2, ,1, 5-HT1B, and 5-HT2A receptor blocking activities. In conclusion, the changes in the position of chloride at phenylpiperazine influenced the serotonergic receptor, adrenoceptor antagonistic activities, but not anti-aggregation and antioxidant activities. Drug Dev Res 71:1,9, 2010. © 2010 Wiley-Liss, Inc. [source]

    Synthesis and Biological Evaluation of 14-Alkoxymorphinans.

    HELVETICA CHIMICA ACTA, Issue 7 2003
    Part 1
    The 14- O -benzylnaltrexones 3,6 were prepared from naltrexone (2) in several steps. The novel compounds were biologically evaluated in radioligand binding and in [35S]GTP,S functional assays in comparison to the reference compound naltrexone. In the binding assay, compounds 3,6 exhibited preference for , opioid receptors, while the parent compound naltrexone shows preference for , receptors. In the functional assay, , antagonist potency of compounds 3,6 was in the range of naltrexone, while , antagonist potency was considerably higher for most novel compounds in comparison to naltrexone. [source]

    Pharmacological profile of essential oils derived from Lavandula angustifolia and Melissa officinalis with anti-agitation properties: focus on ligand-gated channels

    JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2008
    Liping Huang
    Both Melissa officinalis (Mo) and Lavandula angustifolia (La) essential oils have putative anti-agitation properties in humans, indicating common components with a depressant action in the central nervous system. A dual radioligand binding and electrophysiological study, focusing on a range of ligand-gated ion channels, was performed with a chemically validated essential oil derived from La, which has shown clinical benefit in treating agitation. La inhibited [35S] TBPS binding to the rat forebrain gamma aminobutyric acid (GABA)A receptor channel (apparent IC50 = 0.040 ± 0.001 mg mL,1), but had no effect on N -methyl- d -aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or nicotinic acetylcholine receptors. A 50:50 mixture of Mo and La essential oils inhibited [3H] flunitrazepam binding, whereas the individual oils had no significant effect. Electrophysiological analyses with rat cortical primary cultures demonstrated that La reversibly inhibited GABA-induced currents in a concentration-dependent manner (0.01,1 mg mL,1), whereas no inhibition of NMDA- or AMPA-induced currents was noted. La elicited a significant dose-dependent reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission (in contrast to the classic GABAA antagonist picrotoxin which evoked profound epileptiform burst firing in these cells). These properties are similar to those recently reported for Mo. The anti-agitation effects in patients and the depressant effects of La we report in neural membranes in-vitro are unlikely to reflect a sedative interaction with any of the ionotropic receptors examined here. These data suggest that components common to the two oils are worthy of focus to identify the actives underlying the neuronal depressant and anti-agitation activities reported. [source]

    Pharmacological profile of an essential oil derived from Melissa officinalis with anti-agitation properties: focus on ligand-gated channels

    JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 3 2008
    Sawsan Abuhamdah
    A dual radioligand binding and electrophysiological study, focusing on a range of ligand-gated ion channels, was performed with a chemically-validated essential oil derived from Melissa officinalis (MO), which has shown clinical benefit in treating agitation. MO inhibited binding of [35S] t -butylbicyclophosphorothionate (TBPS) to the rat forebrain gamma-aminobutyric acid (GABA)A receptor channel (apparent IC50 0.040±0.001 mg mL,1), but had no effect on N -methyl- d -aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropianate (AMPA) or nicotinic acetylcholine receptors. Electrophysiological analyses with primary cultures of rat cortical neurons demonstrated that MO reversibly inhibited GABA-induced currents in a concentration-dependent manner (0.01,1 mg mL,1), whereas no inhibition of NMDA- or AMPA-induced currents was noted. Interestingly, MO elicited a significant dose-dependent reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission (in contrast to the classical GABAA antagonist picrotoxinin which evoked profound epileptiform burst firing in these cells). The anti-agitation effects in patients and the depressant effects of MO in in-vitro we report in neural membranes are unlikely to reflect a sedative interaction with any of the ionotropic receptors examined here. [source]

    Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia

    JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 4 2006
    C. I. BRANDON
    The aim of the current study was to clone the equine adenosine A2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A -R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A -R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (KD), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p -sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A2A -R agonists revealed that the eA2A -R functionally coupled to G,s as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF- ,B reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF- ,B activity. These results indicate that the heterologously expressed eA2A -R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A -R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease. [source]

    Flavonoids as antagonists at A1 adenosine receptors

    PHYTOTHERAPY RESEARCH, Issue 11 2006
    Stephen P. H. Alexander
    Abstract This study aimed to investigate the potential for ,avonoid action at A1 adenosine receptors in vitro. In a radioligand binding assay for A1 adenosine receptor occupancy in particulate preparations from guinea-pig cerebral cortex, flavonoids competed in concentration-dependent manners with Hill slopes typically not different from unity. Of the ,avonoids tested, quercetin showed highest affinity (pKi value of 5.33). At a concentration of 28 mm, quercetin evoked a rightward shift in the N6 -cyclopentyladenosine-induced inhibition of electrically evoked contractions of the guinea-pig isolated ileum, allowing the calculation of a pKi value of 4.71. These data suggest, therefore, that ,avonoids represent an additional dietary source of A1 adenosine receptor antagonists (beyond the methylxanthines, caffeine and theophylline). Copyright © 2006 John Wiley & Sons, Ltd. [source]

    Systematic interpretation of cyclic nucleotide binding studies using KinetXBase

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2008
    Sonja Schweinsberg
    Abstract Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3,,5,-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users. [source]

    Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo

    BJU INTERNATIONAL, Issue 8 2008
    Peter Ney
    OBJECTIVE To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents. MATERIALS AND METHODS The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1,M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague-Dawley rats, and compared to those of oxybutynin and atropine. RESULTS In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist-stimulated responses in human M1-M5 cell lines and had a similar potency and selectivity profile to the radioligand-binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration-response curve for carbachol with no depression of the maximum, and concentration-dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration-response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume. CONCLUSIONS Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile. [source]

    Adenosine A2A Receptors are Up-regulated in Pick's Disease Frontal Cortex

    BRAIN PATHOLOGY, Issue 4 2006
    José Luís Albasanz PhD
    Adenosine A2A receptors (A2AR) are highly expressed in striatum. However, they are also present in extrastriatal structures. A2AR were studied in post-mortem human frontal cortex from Pick's disease (PiD) and age-matched non-demented controls by radioligand binding assays, Western-blotting, real-time PCR and adenylyl cyclase activity determination. Saturation binding assay using [3H]ZM 241385, a selective A2A antagonist, as radioligand revealed a significant increase in total adenosine A2AR numbers (Bmax) in frontal cortex from PiD samples (191% of control Bmax), suggesting up-regulation of this receptor. A significant increase in the level of A2AR was also detected by Western-blotting. Furthermore, expression of mRNA coding A2AR determined by quantitative real-time PCR was enhanced. In agreement, stimulation of adenylyl cyclase by CGS 21680, a selective A2A receptor agonist, was significantly strengthened. Up-regulation of A2B receptors and their corresponding mRNA was also observed. These results show that A2A adenosine receptor/adenylyl cyclase transduction pathway is up-regulated and sensitized in frontal cortex brain from PiD. [source]

    Identification of domains influencing assembly and ion channel properties in ,7 nicotinic receptor and 5-HT3 receptor subunit chimaeras

    BRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2007
    V J Gee
    Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR ,7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing ,7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on ,7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some ,7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. British Journal of Pharmacology (2007) 152, 501,512; doi:10.1038/sj.bjp.0707429; published online 27 August 2007 [source]

    Sympathectomy reveals ,1A - and ,1D -adrenoceptor components to contractions to noradrenaline in rat vas deferens

    BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2004
    Linda Cleary
    We have previously demonstrated that contractions of rat vas deferens to exogenous noradrenaline involve predominantly ,1A -adrenoceptors, but that contractions to endogenous noradrenaline involve predominantly ,1D -adrenoceptors. In this study, we have examined the effects of sympathectomy on the subtypes of ,1 -adrenoceptor in rat vas deferens in radioligand binding and functional studies. In vehicle-treated tissues, antagonist displacement of [3H]prazosin binding to ,1 -adrenoceptors was consistent with a single population of ,1 -adrenoceptors. Binding affinities for a range of ,1 -adrenoceptor antagonists were expressed as pKi values and correlated with known affinities for ,1 -adrenoceptor subtypes. The correlation was significant only with ,1A -adrenoceptors. In tissues from rats sympathectomised with 6-hydroxy-dopamine (2 × 100 mg kg,1 i.p.), binding affinity for the ,1D -adrenoceptor antagonist BMY 7378 fitted best with a two-site model. In functional studies, the potency of noradrenaline at producing total (phasic plus tonic) but not tonic contractions was increased in tissues from sympathectomised rats. Results obtained from sympathectomised rats suggest that phasic contractions are mainly ,1D -adrenoceptor mediated, whereas tonic contractions are mainly ,1A -adrenoceptor mediated, based on the effects of BMY 7378 and the ,1A -adrenoceptor antagonist RS 100329. It is concluded that the predominant ,1 -adrenoceptor in vehicle-treated rat vas deferens is the ,1A -adrenoceptor, both in terms of ligand binding and contractions to exogenous agonists. The ,1D -adrenoceptor is only detectable by ligand binding following chemical sympathectomy, but is involved in noradrenaline-evoked contractions, particularly phasic contractions, of rat vas deferens. British Journal of Pharmacology (2004) 143, 745,752. doi:10.1038/sj.bjp.0705987 [source]

    Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor

    BRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2004
    Christopher J Langmead
    This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. Specific binding of [3H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein,1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin-A displaying a ,five-fold higher affinity than orexin-B (Ki values of 318±158 and 1516±597 nM, respectively). SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. These studies indicate that [3H]SB-674042 is a specific, high-affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors. British Journal of Pharmacology (2004) 141, 340,346. doi:10.1038/sj.bjp.0705610 [source]

    Competitive MS Binding Assays for Dopamine D2 Receptors Employing Spiperone as a Native Marker

    CHEMBIOCHEM, Issue 10 2005
    Karin V. Niessen
    Abstract A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays. [source]

    Interactions between Conserved Residues in Transmembrane Helices 2 and 7 during Angiotensin AT1 Receptor Activation

    CHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2006
    Gregory V. Nikiforovich
    Site-directed mutagenesis studies and independent molecular modeling studies were combined to investigate the network of inter-residue interactions within the transmembrane region of the angiotensin AT1a receptor. Site-directed mutagenesis was focused on residues Tyr292, Asn294, Asn295, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT1a receptor activation. Replacement of Tyr292 by aspartate in helix 7 abolished radioligand binding to both Y292D and D74Y/Y292D mutant receptors. Reciprocal mutations of Asp74/Asn294, Ser115/Asn294, Ser252/Asn294, and Asn298/Sen115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT1a receptor activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the TM region of the AT1 receptor and the mutants as well as of their complexes with angiotensin II, which were used to rationalize the possible reasons of impairing of function of some mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct strong interaction in the resting conformation of the AT1a receptor. Balance of interactions between residues in helix 2 (as D74) and helix 7 (as N294, N295 and N298) in the AT1 receptors, however, has a crucial role both in determining their functional activity and levels of their expression. [source]

    Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA,

    CHIRALITY, Issue 9 2001
    Tommy N. Johansen
    Abstract We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC50 = 0.025 ,M), low affinity in kainic acid binding (IC50 = 3.6 ,M), and potent AMPA receptor agonist activity on cortical neurons (EC50 = 0.25 ,M), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC50 = 0.039 ,M), but was found to be a relatively weak AMPA receptor agonist (EC50 = 12 ,M). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC50 = 220 ,M). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu2 receptor subtype (KB = 148 ,M). Chirality 13:523,532, 2001. © 2001 Wiley-Liss, Inc. [source]

    SALIVATION TRIGGERED BY PILOCARPINE INVOLVES AQUAPORIN-5 IN NORMAL RATS BUT NOT IN IRRADIATED RATS

    CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2009
    Tetsuya Asari
    SUMMARY 1Using rats, we examined the muscarinic receptor subtype mediating pilocarpine-induced parotid salivary secretion and the contributions of ion transporter systems (effluxes of K+ and Cl - ) and aquaporin-5 (AQP5) translocation to this response in parotid glands in irradiated-induced xerostomia. 2Salivary secretion was significantly lower in irradiated compared with sham-irradiated (normal) rats. In xerostomia rats, 0.4 and 0.8 mg/kg pilocarpine significantly increased parotid salivary secretion, although the salivary volume was still significantly less than in normal rats after the same dose of pilocarpine. 3Pirenzepine (1 × 10,6 to 1 × 10,1 mol/L), AF-DX 116 (3 × 10,6 to 3 × 10,2 mol/L) and N -2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP; 1 × 10,8 to 1 × 10,2 mol/L) dose-dependently displaced radioligand binding to M1, M2 and M3 receptors, respectively, in parotid membranes from both normal and irradiated rats. In each group of rats, 4-DAMP had the highest binding affinity. Pretreatment with 4-DAMP or pirenzepine dose-dependently inhibited pilocarpine-induced parotid secretion in both normal and irradiated rats, with 4-DAMP being markedly more potent than pirenzepine. 4Normal and irradiated-rat parotid cells did not differ significantly in terms of pilocarpine-induced changes in [Ca2+]i, [K+]i and [Cl - ]i. Pilocarpine markedly increased the amount of AQP5 in the apical plasma membrane of parotid cells isolated from normal but not irradiated rats. 5Thus, pilocarpine induces parotid salivary secretion mainly via the M3 receptor subtype in both irradiated and normal rats. The reduction in this pilocarpine-induced secretion seen in irradiated rats is due not to disturbances of intracellular Ca2+ mobilization or ion transporter systems, but rather to a disturbance of AQP5 translocation, which may be involved in the pathogenesis of X-ray irradiation-induced xerostomia. [source]

    HUMAN HEART ,-ADRENOCEPTORS: ,1 -ADRENOCEPTOR DIVERSIFICATION THROUGH ,AFFINITY STATES' AND POLYMORPHISM

    CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2007
    P Molenaar
    SUMMARY 1In atrium and ventricle from failing and non-failing human hearts, activation of ,1 - or ,2 -adrenoceptors causes increases in contractile force, hastening of relaxation, protein kinase A-catalysed phosphorylation of proteins implicated in the hastening of relaxation, phospholamban, troponin I and C-protein, consistent with coupling of both ,1 - and ,2 -adrenoceptors to stimulatory Gsa -protein but not inhibitory Gia -protein. 2Two ,affinity states', namely ,1H and ,1L, of the ,1 -adrenoceptor exist. In human heart, noradrenaline elicits powerful increases in contractile force and hastening of relaxation. These effects are blocked with high affinity by ,-adenoceptor antagonists, including propranolol, (,)-pindolol, (,)-CGP 12177 and carvedilol. Some beta-blockers, typified by (,)-pindolol and (,)-CGP 12177, not only block the receptor, but also activate it, albeit at much higher concentrations (approximately 2 log units) than those required to antagonize the effects of catecholamines. In human heart, both (,)-CGP 12177 and (,)-pindolol increase contractile force and hasten relaxation. However, the involvement of the ,1 -adrenoceptor was not immediately obvious because (,)-pindolol- and (,)-CGP 12177-evoked responses were relatively resistant to blockade by (,)-propranolol. Abrogation of cardiostimulant effects of (,)-CGP 12177 in ,1 -/,2 -adrenoceptor double-knockout mice, but not ,2 -adrenoceptor-knockout mice, revealed an obligatory role of the ,1 -adrenoceptor. On the basis of these results, two ,affinity states' have been designated, the ,1H - and ,1L -adrenoceptor, where the ,1H -adrenoceptor is activated by noradrenaline and blocked with high affinity by beta-blockers and the ,1L -adrenoceptor is activated by drugs such as (,)-CGP 12177 and (,)-pindolol and blocked with low affinity by beta-blockers such as (,)-propranolol. The ,1H - and ,1L -adrenoceptor states are consistent with high- and low-affinity binding sites for (,)-[3H]-CGP 12177 radioligand binding found in cardiac muscle and recombinant ,1 -adrenoceptors. 3There are two common polymorphic locations of the ,1 -adrenoceptor, at amino acids 49 (Ser/Gly) and 389 (Arg/Gly). Their existence has raised several questions, including their role in determining the effectiveness of heart failure treatment with beta-blockers. We have investigated the effect of long-term maximally tolerated carvedilol administration (> 1 year) on left ventricular ejection fraction (LVEF) in patients with non-ischaemic cardiomyopathy (mean left ventricular ejection fraction 23 ± 7%; n = 135 patients). The administration of carvedilol improved LVEF to 37 ± 13% (P < 0.005); however, the improvement was variable, with 32% of patients showing £ 5% improvement. Upon segregation of patients into Arg389Gly-,1 -adrenoceptors, it was found that carvedilol caused a greater increase in left ventricular ejection faction in patients carrying the Arg389 allele with Arg389Arg > Arg389Gly > Gly389Gly. [source]