Abstract Neuronal nicotinic cholinergic receptors mediate the actions of acetylcholine in ganglia and the CNS. These receptors are pentameric proteins formed from combinations of 12 different subunits. Thus, this family of receptors consists of multiple subtypes defined by their subunit composition. All of the receptors are ligand‐gated cation channels that pass sodium, potassium and calcium, but the different subtypes have different biophysical and pharmacological characteristics. These receptors are located on the axons of catecholamine, GABA, acetylcholine and glutamate neurons, so they can influence a large number of diverse functions in the nervous system. In addition to mediating the essential actions of endogenous acetylcholine, these receptors are the primary target of nicotine, the addictive agent in tobacco. Thus, these receptors are directly related to the single most preventable cause of premature morbidity and mortality. In this chapter, we review some of the fundamental aspects of these receptors, with emphasis on the differences in pharmacology among the receptor subtypes and their unusual regulation by exposure to nicotine.
Abstract: Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [ 3 H]tryptamine with two radio‐ligands for monoamine oxidase (MAO), [ 3 H]pargyline, and [ 3 H] 1 ‐methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine ([ 3 H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [ 3 H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [ 3 H]tryptamine binding site. In subcellular distribution studies, [ 3 H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [ 3 H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [ 3 H]tryptamine binding were completely different from their potencies at inhibiting [ 3 H]MPTP binding. Finally, the au‐toradiographic distribution of [ 3 H]tryptamine binding in rat brain v/as different from that of [ 3 H]MPTP and [ 3 H]pargyline. We conclude that the [ 3 H]tryptamine binding site in rat brain is not equivalent to MAO.
We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor α2, α3, or α4 subunits in combination with the β2 or β4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXα2β2, KXα2β4, KXα3β2, KXα3β4, KXα4β2, and KXα4β4. The Kd values of [3H](±)epibatidine ([3H]EB) binding to membranes from these six cell lines ranged from ∼0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [3H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the β2 subunit compared with those containing the β4 subunit. An excellent correlation (r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXα4β2 cells and from rat forebrain tissue, in which [3H]EB binding represents predominantly α4β2 nAChRs. More important, the affinities (Ki values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.
Abstract: Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up‐regulated sites labeled by [ 3 H]cytisine in cerebral cortex appear to be composed exclusively of α4 and β2 subunits. It is unknown whether receptor subtypes that do not bind [ 3 H]‐cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [ 3 H]cytisine with that of [ 3 H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [ 3 H]‐cytisine and [ 3 H]epibatidine. In contrast, no change in [ 3 H]‐epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high‐affinity [ 3 H]cytisine binding. These data indicate that, under the conditions used here, nicotine up‐regulates the α4β2 nicotinic receptor subtype, which can be labeled by [ 3 H]cytisine and [ 3 H]epibatidine, but not non‐α4β2 subtypes, which can be labeled by [ 3 H]epibatidine.
