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Binding Experiments (binding + experiment)
Selected AbstractsMorphine-like substance in leech gangliaFEBS JOURNAL, Issue 8 2000Evidence, immune modulation Binding experiments followed by measurement of nitric oxide release revealed an opiate alkaloid high affinity receptor with no affinity to opioids, representing a new µ-subtype receptor in the brain of the leech Theromyzon tessulatum. In addition, evidence of morphine-like substances was found in immunocytochemical studies and HPLC coupled to electrochemical detection (500 mV and 0.02 Hz). Based on previous evidence of the involvement of morphine as an immune response inhibitor, we demonstrate that in leech ganglia injection of lipopolysaccharide (LPS; a potent immunostimulatory agent derived from bacteria) provoked an increase in the level of ganglionic morphine-like substances after a prolonged latency period of 24 h (from 2.4 ± 1.1 pmol per ganglion to 78 ± 12.3 pmol per ganglion; P < 0.005; LPS injected 1 µg·mL,1); this effect is both concentration- and time-dependent. Finally, we have demonstrated that morphine, after binding to its own receptor, inhibits leech immunocyte activation through adenylate cyclase inhibition and nitric oxide release. This report confirms that morphine is an evolutionarily stable potent immunomodulator. [source] Haptoglobin from psoriatic patients exhibits decreased activity in binding haemoglobin and inhibiting lecithin-cholesterol acyltransferase activityJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 4 2008L Cigliano Abstract Objective, The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of haptoglobin (Hpt). Background, Hpt is a plasma acute-phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free haemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT). Study design, Hpt was isolated from patients with psoriasis vulgaris, and its activity in haemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods, Two affinity chromatography steps, the first using resin-coupled haemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by Enzyme-linked immunosorbent assay with stationary haemoglobin or ApoA-I, Hpt in solution and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results, Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by haemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both haemoglobin binding and LCAT inhibition. Conclusions, In psoriasis, Hpt displays some structure modification(s), which might be associated with the protein function in the disease. [source] Pharmacological characterization of F-180: a selective human V1a vasopressin receptor agonist of high affinityBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2002Miriam Andrés The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V1a receptor subtype (Ki=11 nM) and was selective for this receptor subtype. Functional studies performed on CHO cells expressing human V1a receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (Kact) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V2 and V1b receptor subtypes and an antagonist for the human OT receptor. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V1a receptors, but weak affinity and non selective properties for rat V1a receptors. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. In conclusion, we demonstrate that F-180 is the first selective V1a agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V1a receptor subtypes. British Journal of Pharmacology (2002) 135, 1828,1836; doi:10.1038/sj.bjp.0704634 [source] Research Article: Effective and Specific Inhibition of the CD40,CD154 Costimulatory Interaction by a Naphthalenesulphonic Acid DerivativeCHEMICAL BIOLOGY & DRUG DESIGN, Issue 4 2010Emilio Margolles-Clark Costimulatory interactions are important regulators of T-cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small-molecule inhibitors of the CD40,CD154 costimulatory protein,protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration-dependently inhibit the CD154(CD40L)-induced cellular responses in human THP-1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40,CD154 interaction with sub-micromolar activity, but it also has considerably more than 100-fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF-R1,TNF-,, BAFF-R(CD268),BAFF(CD257/BLys), OX40(CD134),OX40L(CD252), RANK(CD265),RANKL(CD254/TRANCE), or 4-1BB(CD137),4-1BBL. There is now sufficient structure-activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction. [source] Molecular imprinted solid-phase extraction of huperzine A from Huperzia SerrataJOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2009Guosong Wang Abstract On the basis of the non-covalent interaction between template and monomer, porous molecularly imprinted polymers (MIPs) were synthesized by a thermal-initiated polymerization method using huperzine A as template, acrylamide, or methacrylic acid as function monomer, ethylene glycol dimethacrylate as cross-linking agent. The interaction between template and functional monomers was studied by UV spectrophotometry, which showed a formation of huperzine A-monomer complexes with stoichiometric ratio of 1 : 2 in the pre-polymerized systems. The resultant MIP particles were tested in the equilibrium binding experiment to analyze their adsorption ability to huperzine A, and were characterized by Fourier Transform Infrared (FTIR) study. The recognition properties of MIP were estimated in solid-phase extraction by selecting four compounds (isolated from the Chinese herb Huperzia serrata) as substrates, and were compared with and prior to those of the NIP. High affinity and adsorption of MIP1 which was prepared in chloroform with huperzine A as imprinted molecule, and acrylamide (AM) as functional monomer, made an attractive application of MIP1 in separation processes. In final, using MIP1 solid-phase extraction micro-column, huperzine A was enriched and separated from the real extraction sample of Huperzia serrata. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [source] Analyzing ligand depletion in a saturation equilibrium binding experimentBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2006Enrique Claro Abstract I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [3H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high density of muscarinic receptors. These features allow the instructor to devote especial emphasis to evaluate ligand depletion, and therefore, stress the subtle but fundamental difference between total (added) ligand and free ligand concentration at equilibrium. [source] Protein kinases A and C stimulate the Na+ active transport in frog skeletal muscle without an appreciable change in the number of sarcolemmal Na+ pumpsACTA PHYSIOLOGICA, Issue 4 2005R. A. Venosa Abstract Aim:, The activation of both protein kinases A (PKA) and protein kinases C (PKC) in some cell types increases and in others reduces active Na+ efflux. These effects have been ascribed to either a change in the rate of ionic translocation by a fixed number of Na+ pumps or, a change in the number of plasma membrane pumps. The purpose of the present experiments was to study the effect of activating PKA and PKC on the Na+ extrusion by the Na+ pump in frog skeletal muscle. Methods:, Na+ (22Na+) fluxes and ouabain (3H-ouabain) binding were measured in frog sartorius muscles. Results:, Both activation of PKA and PKC increased the active Na+ extrusion by a factor of two; these effects were not additive. Ouabain binding experiments indicated that the pump stimulation by activation of these kinases is not associated with any significant increase in the number of plasma membrane pumps. Stimulation of the active Na+ efflux by protein kinase activation (no change in the number of sarcolemmal pumps) and by hypotonicity (increase in the number of pumps) could be elicited in the same preparation and they were additive. Conclusion:, It is concluded that in frog skeletal muscle fibres, (1) activation of both PKA and PKC stimulate the Na+ pump by increasing its rate of ionic translocation; and (2) two modes of Na+ active transport (with and without an increase in the number of pumps) are operative, and can be at work simultaneously, a phenomenon to be reckoned with. [source] Novel estrogen receptor ligands and their structure,activity relationship evaluated by scintillation proximity assay for high-throughput screeningDRUG DEVELOPMENT RESEARCH, Issue 4 2005Ling 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] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pHFEBS JOURNAL, Issue 10 2003Gregorio Fernández-Ballester The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ,G = 4.0 ± 0.2 kcal·mol,1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7,9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5,4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family. [source] Preparation and Characteristics of Esculin-Imprinted PolymersHELVETICA CHIMICA ACTA, Issue 6 2007Guo-Song Wang Abstract Four molecularly imprinted polymers (MIPs) were prepared in MeOH with esculin (=6,7-dihydroxycoumarin 6-(, - D -glucopyranoside)=6-(, - D -glucopyranosyloxy)-7-hydroxy-2H -1-benzopyran-2-one) as the imprinted molecule, methacrylic acid (=2-methylprop-2-enoic acid; MAA), acrylamide (=prop-2-enamide; AM), 4-vinylpyridine (=4-ethenylpyridine; 4-VP), or 2-vinylpyridine (=2-ethenylpyridine; 2-VP) as the functional monomer, respectively, as well as ethylene glycol dimethacrylate (=2-methylprop-2-enoic acid ethane-1,2-diyl ester; EGDMA) as the cross-linking agent. The interaction between the template and the functional monomers was investigated by fluorescence and UV spectrophotometry, respectively, which revealed the presence of esculin/monomer complexes in the stoichiometric ratio 1,:,2 in the pre-polymerization mixture. The resultant polymers were studied in equilibrium binding experiments to evaluate the recognition ability and the binding capacity towards esculin. The results showed that MIP1, prepared with MAA as the functional monomer, exhibited advantageous characteristics of high binding capacity, optimal imprinting effect, and good selectivity towards esculin. The Scatchard analysis indicated that there are two types of binding sites in MIP1, and its binding parameters including the apparent maximum numbers of binding sites and the dissociation constants were calculated. Finally, by packing an SPE column (SPE=solid-phase extraction) with MIP1, the esculin was separated and enriched successfully by this sorbent from samples of Cortex fraxini, and the average recovery was up to 74.7%. [source] Characterization of nociceptin binding sites by novel peptide analogs and radioprobesJOURNAL OF NEUROCHEMISTRY, Issue 2003S. Benyhe A number of new synthetic nociceptin ligands were studied in receptor binding and functional tests in rat brain membranes and in cloned systems. Ligand binding experiments were performed with three different radioprobes developed in our lab. The nociceptin derivatives exhibited high affinity in competition experiments. Receptor-mediated G-protein activation was determined in [35S]GTPgS binding assays. Among the new structures examined, Ac-RYYRIK-ol was found to be only a weak stimulator by itself, whereas this compound inhibited receptor-mediated G-protein activation. These data suggest that Ac-RYYRIK-ol is a high affinity peptide antagonist for the nociceptin receptor. Acknowledgements:, Supported by the Hungarian Scientific Research Fund OTKA T-035211, T-033078, T-030841, and the Ministry of Education, NKFP 1/027 Hungary. [source] Competition STD NMR for the detection of high-affinity ligands and NMR-based screeningMAGNETIC RESONANCE IN CHEMISTRY, Issue 6 2004Yu-Sen Wang Abstract The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (,-site amyloid precursor protein cleaving enzyme 1) protein,inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure,activity relationships, which are used to optimize these NMR hits into viable drug leads. Copyright © 2004 John Wiley & Sons, Ltd. [source] Tryptophan as a three-way switch in regulating the function of the secretory signalling glycoprotein (SPS-40) from mammary glands: structure of SPS-40 complexed with 2-methylpentane-2,4-diol at 1.6,Ĺ resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2009Pradeep Sharma The 40,kDa secretory signalling glycoprotein (SPS-40) is the first example with Trp78 in three functional orientations: (i) a resting state with a pinched conformation, (ii) a stacked conformation when bound to hexasaccharide and (iii) an obstructive conformation when inhibited by 2-methylpentane-2,4-diol (MPD). Trp78 is present in the core of the sugar-binding groove. The hexasaccharide N -acetylglucosamine (GlcNAc6) has been shown to bind to SPS-40. As a result of this, the conformation of Trp78 alters from the native pinched conformation (,1 = ,65.5°, ,2,1 = ,78.8°, ,2,2 = 97.5°) to the stacked conformation (,1 = ,170.0°, ,2,1 = ,114.3°, ,2,2 = 61.6°). Further binding experiments showed that saccharide binding does not occur in the presence of 20% MPD. The crystal structure determination of the complex of SPS-40 with MPD revealed the presence of two MPD molecules in the sugar-binding groove. The very tightly bound MPD molecules at subsites ,2 and ,1 induced an unexpected and a rarely observed conformation of Trp78 (,1 = 55.9°, ,2,1 = 90.2°, ,2,2 = ,88.9°) which is termed an obstructive conformation. The binding of MPD molecules also twisted the side chains of Glu269 and Ile272 considerably. These residues are also part of the sugar-binding groove. The observed obstructive conformation of the side chain of Trp78 in the present structure is the exact opposite of the stacked conformation. This rarely observed conformation is stabilized by a number of hydrogen bonds between Trp78 and Asn79 through water molecules W49, W229, W269, W547 and W557. [source] The ,-adrenoceptor antagonist, zolertine, inhibits ,1D- and ,1A-adrenoceptor-mediated vasoconstriction in vitroAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2000M. Ibarra 1 The antagonist effect of zolertine (4-phenyl-1-[2-(5-tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (,1D-adrenoceptors), mesenteric (,1A/D-adrenoceptors) and caudal arteries (,1A-adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (,1B-adrenoceptors), was investigated in endothelium-denuded arterial rings. 2 The selective ,1D-adrenoceptor agonist, noradrenaline, elicited concentration-dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid=aorta>>.Gt;mesenteric=rabbit aorta>caudal arteries. 3 The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48±0.18; SHR, 7.43±0.13 and WKY, 7.57±0.24; SHR, 7.40±0.08, respectively. Zolertine was a non-competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98±0.16; SHR, 6.81±0.18 in the mesenteric artery, WKY, 5.73±0.11; SHR, 5.87±0.25 in the caudal artery and 6.65±0.09 in rabbit aorta. 4 Competition binding experiments using the ,1-adrenoceptor antagonist [3H]prazosin showed a zolertine pKi of 6.81±0.02 in rat liver (,1B-adrenoceptors) and 6.35±0.04 in rabbit liver (,1A-adrenoceptors) membranes. 5 Zolertine showed higher affinity for ,1D-adrenoceptors compared to ,1A-adrenoceptors, while it had an intermediate affinity for ,1B-adrenoceptors. The ability of the ,1-adrenoceptor antagonist zolertine to block ,1D-adrenoceptor-mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency. [source] Microseparation techniques for the study of the enantioselectivity of drug,plasma protein bindingBIOMEDICAL CHROMATOGRAPHY, Issue 3 2009Laura Escuder-Gilabert Abstract Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer,protein interactions, enantiomer,enantiomer interactions as well as chiral drug,drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug,plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd. [source] In vivo saturation binding of GABA-A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometryBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2009Seth C. Hopkins Abstract Typically, the dose-occupancy curves for GABA-A receptor ligands are determined using in vivo binding of [3H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA-A ligands zolpidem, (RS)-zopiclone, L-838417 and flumazenil, to demonstrate affinity and saturation of the filter-retained, membrane-bound fraction. The in vivo binding of flumazenil and L-838417 both saturated around 200,nm, at a similar level to the specific binding of (S)-zopiclone after doses of the racemic zopiclone, using (R)-zopiclone to estimate non-specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA-A receptors in vivo (200,nm). Dose-occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)-isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright © 2009 John Wiley & Sons, Ltd. [source] Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytesBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2010K Varani Background and purpose:, Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. Experimental approach:, mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-,B activation, tumour necrosis factor , (TNF-,) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. Key results:, mRNA and protein for A1, A2A, A2B and A3 adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A2A and A3 adenosine receptors inhibited p38 MAPK and NF-,B pathways, an effect abolished by selective adenosine antagonists. A2A and A3 receptor agonists decreased TNF-, and IL-8 production. The phosphoinositide 3-kinase or Gs pathways were involved in the functional responses of A3 or A2A adenosine receptors. Synoviocyte A1 and A2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A2A and A3 adenosine receptors was closely associated with a down-regulation of the inflammatory status. Conclusions and implications:, These results indicate that A2A and A3 adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation. [source] Binding of GTP,[35S] is regulated by GDP and receptor activation.BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2010Studies with the nociceptin/orphanin FQ receptor Background and purpose:, We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5,-[,-thio]triphosphate (GTP,S) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-coupled G-proteins. Experimental approach:, In GTP,[35S] binding experiments, using stable (CHOhNOP) and inducible (CHOINDhNOP) recombinant human and rat NOP we have measured: (i) ligand-specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTP,S association kinetics. Key results:, GTP,S competition curves were shallow and modelled by high- and low-affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 µM N/OFQ a high-affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy-dependent manner, the partial agonists [F/G]N/OFQ(1-13)NH2 ([Phe1,(CH2 -NH)Gly2]-nociceptin(1-13)NH2) and naloxone benzoylhydrazone both reduced the fraction of high-affinity sites for GDP (relative to basal). While the pIC50 for high-affinity GDP binding site did not decrease in the presence of 1 µM N/OFQ, N/OFQ produced a significant reduction in pIC50 for the low-affinity site. Agonist-mediated decrease in affinity for GDP binding was efficacy-dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy-dependent), present during receptor activation representing the majority of binding. Conclusions and implications:, The affinity of GTP,[35S] was regulated by GDP and receptor activation caused increased binding of GTP,[35S] through a reduction in GDP affinity. [source] Competitive MS Binding Assays for Dopamine D2 Receptors Employing Spiperone as a Native MarkerCHEMBIOCHEM, Issue 10 2005Karin 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] | ||||||||||||||||