High affinity binding of [3H]epibatidine to rat brain membranes

Abstract

Several compounds, such as epibatidine, A-85380, and their analogs, have been identified recently as nAChR ligands whose affinities lie in the low picomolar range. Accurate measurement of such high affinities is fraught with certain technical difficulties, which may account for the inconsistency of previously reported affinities of epibatidine, ranging from 4 to 60 pM. Here, we demonstrate that (+/-)-[3H]epibatidine (1-500 pM) binds to a single population of sites in rat brain with KD of 8 +/- 2 pM. This affinity was confirmed in both kinetic experiments and competition assays with (+/-)-[3H]epibatidine and (-)-[3H]cytisine, which were performed under experimental conditions developed specifically for ligands with subnanomolar affinities. Variations from these conditions decreased the observed affinities.

Nicotine self-administration in rats: estrous cycle effects, sex differences and nicotinic receptor binding

“…Different subtypes of nAChRs differ substantially in the rate of, and recovery from, desensitization (Alkondon and Albuquerque 1993;Vibat et al 1995;Hsu et al 1996;Fenster et al 1997;Ke et al 1998;Fryer et al 1999). Since [ 3 H]epibatidine is capable of binding to multiple receptor subtypes (Gerzanich et al 1995;Houghtling et al 1995;Marks et al 1998;Gnadisch et al 1999) the importance of changes in individual subunits cannot be determined. The nicotine exposure resulting from self-administration may have only partially desensitized some or all of these subypes.…”

Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation

“…For our primary screen we measured binding displacement activity of an extract versus the radioligand [3H]epibatidine. This radioligand was chosen for its high selectivity for nicotinic receptors but (for our purposes) approximately equal high affinity for most nicAChR subtypes (Brioni et al, 1997;Gerzanich et al, 1995;Gradisch et al, 1999). As above, we first determined the concentration or dilution of plant extract required to block 50% of the specific bound [3H]epibatidine in rat brain homogenates.…”

Nicotinic Agonists, Antagonists, and Modulators From Natural Sources

“…Binding data for several agonists and antagonists versus [ 3 H]nicotine, [ 3 H]cytisine and [ 3 H]N-methylcarbamylcholine with brain membranes gave virtually identical K i values versus each agent (Anderson and Arneric, 1994). Binding data for six agonists versus [ 3 H]epibatidine or [ 3 H]cytisine with brain membranes also correlated well (Gnädisch et al, 1999), as did K i values in a comparison of ten agonists versus either [ 3 H]ABT-418 or [ 3 H]cytisine . In a limited comparison the K i for (−)-nicotine was nearly eightfold greater versus [ 3 H]epibatidine than versus [ 3 H]nicotine (Beach et al, 1998).…”