Studies of neuronal nicotinic acetylcholine receptors in the rat pineal gland: A model for native receptors composed of alpha-3 and beta-4 subunits

Neuronal nicotinic acetylcholine receptors (nAChRs) are located throughout the central and peripheral nervous systems. They are ligand-gated ion channels composed of alpha and beta subunits. To date, nine alpha (2--10) and three beta (2--4) subunit genes have been identified. Due to the presence of multiple subunits, there are many possible combinations that can form functional receptor subtypes, each displaying distinct pharmacological properties.; Studies of nAChRs in heterologous expression systems have confirmed receptor diversity and provided important information on the pharmacology, function, and regulation of defined receptor subtypes. However, the precise subtypes of nAChRs in native tissues are less understood. Identifying the subunit composition of native nAChRs is important to establishing the physiological roles played by different subtypes in vivo.; The presence of nAChRs in the rat pineal gland was previously suggested by immunocytochemistry and autoradiographic studies. Furthermore, the expression of mRNA encoding for the alpha3, beta2, and beta4 subunits has been demonstrated. The objectives of this research were to assess the pharmacology, subunit composition, and regulation of nAChRs in the rat pineal.; Radioligand binding experiments indicated the pineal gland expresses a high density of [3H]EB labeled binding sites with a K d of 103 pM. Nicotinic drugs competed for [3H]EB binding sites with a rank order of A85380 > cytisine > nicotine > DHBE. In parallel experiments using alpha3beta4 HEK cells, the rank order of potencies, and absolute Ki values were similar to those measured in the rat pineal.; We utilized whole-cell patch clamp to study the functional pharmacology of nAChRs in dissociated rat pinealocytes. The rank order of nicotinic agonists' affinities for current activation was A85380 > DNWP > cytisine ∼ nicotine > acetylcholine. In terms of drug efficacy, cytisine was equally efficacious to nicotine and acetylcholine in activating current. The antagonists, mecamylamine, curare, and DHbetaE inhibited nicotine-activated currents, but MLA and alpha-Bgtx, were ineffective.; Immunoprecipitation with subunit selective antibodies raised against the alpha3, beta2, and beta4 subunits was performed to identify subunit associations of nAChRs from rat pineal. Immunoprecipitation of virtually all [3H]EB labeled receptors was seen for the anti-alpha3 and anti-beta4 antibodies but not the anti-beta2 antibody. Furthermore, initial incubation with anti-alpha3 or anti-beta4 antibodies eliminated virtually all specific immunoprecipitation by a second incubation with either the anti-alpha3 or anti-beta4, indicating the alpha3 and beta4 subunits are associated in the majority of nAChRs in the rat pineal. Additionally, the alpha3beta4 receptor subtype appeared to decrease following chronic treatment with the cholinesterase inhibitor, DFP.; Our findings indicate the rat pineal gland serves as an excellent model in which to study the pharmacology and functional roles of native nAChR containing the alpha3 and beta4 subunit combination.