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High-affinity Site (high-affinity + site)
Selected AbstractsPharmacological characterization of cis -nitromethylene neonicotinoids in relation to imidacloprid binding sites in the brown planthopper, Nilaparvata lugensINSECT MOLECULAR BIOLOGY, Issue 1 2010X. Xu Abstract Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) and extensively used in areas of crop protection and animal health to control a variety of insect pest species. Here we describe that two cis -nitromethylene neonicotinoids (IPPA152002 and IPPA152004), recently synthesized in our laboratory, discriminated between the high and low affinity imidacloprid binding sites in the brown planthopper, Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. [3H]imidacloprid has two binding sites with different affinities (Kd value of 0.0035 ± 0.0006 nM for the high-affinity site and 1.47 ± 0.22 nM for the low-affinity site). Although the cis -nitromethylene neonicotinoids showed low displacement ability (Ki values of 0.15 ± 0.03 µM and 0.42 ± 0.07 µM for IPPA152002 and IPPA152004, respectively) against [3H]imidacloprid binding, low concentrations (0.01 µM) of IPPA152002 completely inhibited [3H]imidacloprid binding at its high-affinity site. In Xenopus oocytes co-injected with cRNA encoding Nl,1 and rat ,2 subunits, obvious inward currents were detected in response to applications of IPPA152002 and IPPA152004, although the agonist potency is reduced to that of imidacloprid. The previously identified Y151S mutation in Nl,1 showed significant effects on the agonist potency of IPPA152002 and IPPA152004, such as a 75.8% and 70.6% reduction in Imax, and a 2.4- and 2.1-fold increase in EC50. This data clearly shows that the two newly described cis -nitromethylene neonicotinoids act on insect nAChRs and like imidacloprid, discriminated between high and low affinity binding sites in N. lugens native nAChRs. These compounds may be useful tools to further elucidate the pharmacology and nature of neonicotinoid binding sites. [source] ,-Hydroxybutyrate binds to the synaptic site recognizing succinate monocarboxylate: A new hypothesis on astrocyte,neuron interaction via the protonation of succinateJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2008Tünde Molnár Abstract Succinate (SUC), a citrate (CIT) cycle intermediate, and carbenoxolone (CBX), a gap junction inhibitor, were shown to displace [3H],-hydroxybutyrate ([3H]GHB), which is specifically bound to sites present in synaptic membrane subcellular fractions of the rat forebrain and the human nucleus accumbens. Elaboration on previous work revealed that acidic pH-induced specific binding of [3H]SUC occurs, and it has been shown to have a biphasic displacement profile distinguishing high-affinity (Ki,SUC = 9.1 ± 1.7 ,M) and low-affinity (Ki,SUC = 15 ± 7 mM) binding. Both high- and low- affinity sites were characterized by the binding of GHB (Ki,GHB = 3.9 ± 0.5 ,M and Ki,GHB = 5.0 ± 2.0 mM) and lactate (LAC; Ki,LAC = 3.9 ± 0.5 ,M and Ki,LAC = 7.7 ± 0.9 mM). Ligands, including the hemiester ethyl-hemi-SUC, and the gap junction inhibitors flufenamate, CBX, and the GHB binding site-selective NCS-382 interacted with the high-affinity site (in ,M: Ki,EHS = 17 ± 5, Ki,FFA = 24 ± 13, Ki,CBX = 28 ± 9, Ki,NCS-382 = 0.8 ± 0.1 ,M). Binding of the Na+,K+ -ATPase inhibitor ouabain, the proton-coupled monocarboxylate transporter (MCT)-specific ,-cyano-hydroxycinnamic acid (CHC), and CIT characterized the low-affinity SUC binding site (in mM: Ki,ouabain = 0.13 ± 0.05, Ki,CHC = 0.32 ± 0.07, Ki,CIT = 0.79 ± 0.20). All tested compounds inhibited [3H]SUC binding in the human nucleus accumbens and had Ki values similar to those observed in the rat forebrain. The binding process can clearly be recognized as different from synaptic and mitochondrial uptake or astrocytic release of SUC, GHB, and/or CIT by its unique GHB selectivity. The transient decrease of extracellular SUC observed during epileptiform activity suggested that the function of the synaptic target recognizing protonated succinate monocarboxylate may vary under different (patho)physiological conditions. Furthermore, we put forward a hypothesis on the synaptic activity-regulated signaling between astrocytes and neurons via SUC protonation. © 2008 Wiley-Liss, Inc. [source] Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2004Christopher 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] One-pot synthesis of surface-functionalized molecularly imprinted polymer microspheres by iniferter-induced "living" radical precipitation polymerizationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 15 2010Junyi Li Abstract This article describes for the first time the development of a new polymerization technique by introducing iniferter-induced "living" radical polymerization mechanism into precipitation polymerization and its application in the molecular imprinting field. The resulting iniferter-induced "living" radical precipitation polymerization (ILRPP) has proven to be an effective approach for generating not only narrow disperse poly(ethylene glycol dimethacrylate) microspheres but also molecularly imprinted polymer (MIP) microspheres with obvious molecular imprinting effects towards the template (a herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)), rather fast template rebinding kinetics, and appreciable selectivity over structurally related compounds. The binding association constant Ka and apparent maximum number Nmax for the high-affinity sites of the 2,4-D imprinted polymer were determined by Scatchard analysis and found to be 1.18 × 104 M,1 and 4.37 ,mol/g, respectively. In addition, the general applicability of ILRPP in molecular imprinting was also confirmed by the successful preparation of MIP microspheres with another template (2-chloromandelic acid). In particular, the living nature of ILRPP makes it highly useful for the facile one-pot synthesis of functional polymer/MIP microspheres with surface-bound iniferter groups, which allows their direct controlled surface modification via surface-initiated iniferter polymerization and is thus of great potential in preparing advanced polymer/MIP materials. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3217,3228, 2010 [source] Preparation of molecularly imprinted polymer microspheres via atom transfer radical precipitation polymerizationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 13 2009Baiyi Zu Abstract The first combined use of atom transfer radical polymerization (ATRP) and precipitation polymerization in the molecular imprinting field is described. The utilized polymerization technique, namely atom transfer radical precipitation polymerization (ATRPP), provides MIP microspheres with obvious molecular imprinting effects towards the template, fast template binding kinetics and an appreciable selectivity over structurally related compounds. The living chain propagation mechanism in ATRPP results in MIP spherical particles with diameters (number-average diameter Dn , 3 ,m) much larger than those prepared via traditional radical precipitation polymerization (TRPP). In addition, the MIP microspheres prepared via ATRPP have also proven to show significantly higher high-affinity binding site densities on their surfaces than the MIP generated via TRPP, while the binding association constants Ka and apparent maximum numbers Nmax of the high-affinity sites as well as the specific template bindings are almost the same in the two cases. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3257,3270, 2009 [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] | ||||||||||