Abstract

The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [³H]epibatidine ([³H]EB) binds with an affinity of ∼100 pM to nAChRs in the pineal gland, and the density of these sites is ∼5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at α3β4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block ⁸⁶Rb⁺ efflux in the cells expressing heterologous α3β4 nAChRs. Measurements of mRNA indicated the presence of transcripts for α3, β2, and β4 nAChR subunits but not those for α2, α4, α5, α6, α7, or β3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [³H]EB-labeled nAChRs contained α3 and β4 subunits associated in one complex. The β2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the α3β4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.