UB-165: A novel nicotinic agonist with subtype selectivity implicates the alpha 4 beta 2*subtype in the modulation of dopamine release from rat striatal synaptosomes

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha 3 beta 2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha 4 and beta 2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha 4 beta 2* and alpha 3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha 7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [H-3]-dopamine release from striatal synaptosomes with EC50 values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)- UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of Rb-86(+) efflux from thalamic synaptosomes, a model of an alpha 4 beta 2* nAChR response, (+/-)- UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha 4 beta 2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)- UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca2+ in SH-SY5Y cells, a functional assay for native alpha 3-containing nAChR. These data support the involvement of alpha 4 beta 2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.