Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors (original) (raw)
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The unusual nature of epibatidine responses at the α4β2 nicotinic acetylcholine receptor
Neuropharmacology, 2000
The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat α4β2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the α4β2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat α4β2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.
Epibatidine: Impact on Nicotinic Receptor Research
Cellular and Molecular Neurobiology, 2003
Epibatidine is a natural product that was isolated and identified by Daly and coworkers in 1992. Since that time, it has had a profound influence on the investigation of a 4ß 2 nicotinic cholinergic (nACh) receptor pharmacophore models, and has inspired the development of novel agents with therapeutic potential in CNS disorders. Apart from acetylcholine and nicotine, probably no other
Epibatidine Binds with Unique Site and State Selectivity to Muscle Nicotinic Acetylcholine Receptors
Journal of Biological Chemistry, 1998
Ligand binding sites in fetal (␣ 2 ␥␦) and adult (␣ 2 ␦⑀) muscle acetylcholine receptors are formed by ␣␦, ␣␥, or ␣⑀ subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the ␦, ␥, or ⑀ subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to ␣␥, ␣␦, and ␣⑀ intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is ␣⑀ > ␣␥ > ␣␦, whereas for carbamylcholine the rank order is ␣␦ Х ␣⑀ > ␣␥. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunitomitted receptors (␣ 2 ␦ 2 , ␣ 2 ␥ 2 , and ␣ 2 ⑀ 2) reveal that in the desensitized state, the ␣␦ interface forms the low affinity epibatidine site, whereas the ␣␥ and ␣⑀ interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.
Journal of Pharmacology and Experimental Therapeutics, 2004
Human nicotinic acetylcholine receptor (nAChR) ␣7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows ␣7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125 Ilabeled ␣-bungarotoxin (I-Bgt) binding sites corresponding to human ␣7 (h␣7)-nAChRs on the surface of most cells. I-Bgt binds to h␣7-nAChRs in membrane fractions with a typical apparent K D value of ϳ5 nM and B max value of ϳ1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h␣7-nAChRs is evident as rapid, transient inward current responses to (Ϫ)-nicotine. Nicotine treatment of transfected cells produces dose-and time-dependent increases (up to ϳ100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for Portions of this work were presented previously [Peng J-H, Leonard SS, and Lukas RJ (1998) Heterologous expression of epibatidine-and ␣-bungarotoxinbinding human ␣7-nicotinic acetylcholine receptor in a native receptor-null human epithelial cell line. Soc Neurosci Abstr 24:831].
European Journal of Medicinal Chemistry, 2006
Neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane ligand-gated ion channels. Recent research demonstrated that selective nAChR ligands may have therapeutic potential in a number of CNS diseases and disorders. The alkaloid epibatidine is a highly potent nonopioid analgesic and nAChR agonist, but too toxic to be a useful ligand. To develop ligands selective for distinct nAChR subtypes and with reduced toxicity, a series of epibatidine and homoepibatidine analogues were synthesized. (±)-8-Methyl-3-(pyridin-3-yl)-8-azabicyclo[3,2,1]oct-2-ene, showed high affinity towards α4β2 (K i = 2 nM), subtype selectivity (α4β2/α7 affinity ratio > 100) and relatively low toxicity in mice and can be labeled with 11 C and 18 F as positron emission tomography (PET) tracers for imaging of nAChRs.
Journal of Pharmacology and Experimental Therapeutics, 2005
Human nicotinic acetylcholine receptor (nAChR) ␣7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows ␣7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125 Ilabeled ␣-bungarotoxin (I-Bgt) binding sites corresponding to human ␣7 (h␣7)-nAChRs on the surface of most cells. I-Bgt binds to h␣7-nAChRs in membrane fractions with a typical apparent K D value of ϳ5 nM and B max value of ϳ1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h␣7-nAChRs is evident as rapid, transient inward current responses to (Ϫ)-nicotine. Nicotine treatment of transfected cells produces dose-and time-dependent increases (up to ϳ100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for Portions of this work were presented previously [Peng J-H, Leonard SS, and Lukas RJ (1998) Heterologous expression of epibatidine-and ␣-bungarotoxinbinding human ␣7-nicotinic acetylcholine receptor in a native receptor-null human epithelial cell line. Soc Neurosci Abstr 24:831].
Journal of Pharmacology and Experimental Therapeutics, 2004
Human nicotinic acetylcholine receptor (nAChR) ␣7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows ␣7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125 Ilabeled ␣-bungarotoxin (I-Bgt) binding sites corresponding to human ␣7 (h␣7)-nAChRs on the surface of most cells. I-Bgt binds to h␣7-nAChRs in membrane fractions with a typical apparent K D value of ϳ5 nM and B max value of ϳ1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h␣7-nAChRs is evident as rapid, transient inward current responses to (Ϫ)-nicotine. Nicotine treatment of transfected cells produces dose-and time-dependent increases (up to ϳ100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for
Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor
Biochemical and Biophysical Research Communications, 2008
The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabelled ligand binding techniques, we show that the binding of [ 3 H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 µM. These classes of sites exist in approximately equal numbers and all [ 3 H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.
RSC Advances, 2013
The highly potent natural alkaloid epibatidine remains a source of inspiration in the search for new analgesic drugs. In this paper, we describe an expansion of our previously reported synthesis of epibatidine analogues, and five synthetic alkaloids characterized by a symmetric, 1-substituted 7-azabicyclo[2.2.1]heptane skeleton, were evaluated for their biological activity. Two of these are binding selectively to the a 4 b 2 subtype of the nicotinic acetylcholine receptor. Their K i values were determined to be 40 and 290 nM. After a favourable evaluation of these compounds' cytotoxicity and metabolic stability, they were submitted to a rat tail flick test. The compounds did not show antinociceptive effects, which may be caused by a combination of insufficient potency and poor brain penetration.
Identification of a novel nicotinic binding site in mouse brain using [ 125 I]-epibatidine
British Journal of Pharmacology, 2000
Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high anity. In this study, [ 125 I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. 2 Binding of [ 125 I]-epibatidine was saturable and apparently monophasic in each brain region (K D =71+12 pM mean+s.e.mean across regions) but inhibition of [ 125 I]-epibatidine binding (200 pM) by A85380, cytisine and (7)-nicotine was biphasic, indicating the presence of multiple binding sites. 3 The sites with lower agonist anity comprised 30.0+2.2, 58.6+0.1 and 48.7+3.3% of speci®c [ 125 I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. 4 The anity dierence between A85380-sensitive and-resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to dierentiate agonist-sensitive and-resistant sites. 5 The pharmacological pro®les of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and ®ve antagonists. The pro®les were indistinguishable across regions, implying that A85380-resistant [ 125 I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. 6 The pharmacological pro®le of the A85380-resistant sites is very dierent from that previously reported for high anity (7)-[ 3 H]-nicotine-, [ 125 I]-a-bungarotoxin-, or [ 125 I]-a-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.