Tritiated acetylcholine was used to measure binding sites with characteristics of nicotinic cholinergic receptors in rat brain. Regulation of the binding sites in vivo was examined by administering two drugs that stimulate nicotinic receptors directly or indirectly. After 10 days of exposure to the cholinesterase inhibitor diisopropyl fluorophosphate, binding of tritiated acetylcholine in the cerebral cortex was decreased. However, after repeated administration of nicotine for 10 days, binding of tritiated acetylcholine in the cortex was increased. Saturation analysis of tritiated acetylcholine binding in the cortices of rats treated with diisopropyl fluorophosphate or nicotine indicated that the number of binding sites decreased and increased, respectively, while the affinity of the sites was unaltered.

Although the predominant subtype in rat brain was a4b2, non-a4b2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, a3b4-like binding accounted for more than 40% of [ 125 I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in a3b4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in a3b2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.

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We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor ␣2, ␣3, or ␣4 subunits in combination with the ␤2 or ␤4 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 KX␣2␤2, KX␣2␤4, KX␣3␤2, KX␣3␤4, KX␣4␤2, andbinding to membranes from these six cell lines ranged from ϳ0.02 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 [ 3 H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the ␤2 subunit compared with those containing the ␤4 subunit. An excellent correlation (r Ͼ 0.99) of the binding affinities of the 10 agonists was observed between receptors from KX␣4␤2 cells and from rat forebrain tissue, in which [3 H]EB binding represents predominantly ␣4␤2 nAChRs. More important, the affinities (K i 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 (Ϫ)-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.Neuronal nicotinic acetylcholine receptors (nAChRs) are found throughout the CNS and peripheral nervous system. In the CNS, they are located on neuronal cell bodies and dendrites, where they mediate fast excitatory postsynaptic responses to acetylcholine and other nicotinic agonists, as well as on axon terminals or preterminal sites. These receptors are often found associated with dopamine, norepinephrine, GABA, acetylcholine, and glutamate neurons, where they can mediate the influence of acetylcholine and other nicotinic agonists on the firing of these neurons and the release of their transmitters. In the peripheral nervous system, nAChRs mediate excitatory neurotransmission at virtually all autonomic ganglia, the adrenal gland, and at sensory ganglia; therefore, they are critical for the normal function and adaptation of nearly all organ systems. In addition, these receptors are found in the pineal gland (Marks et al., 1998;Hernandez et al., 1999;Perry et al., 2002), where they may influence melatonin secretion (Yamada et al., 1998), vascular endothelial and bronchial epithelial cells Maus et al., 1998), and skin keratinocytes (Grando et al., 1995), where their functions are not yet known.nAChRs are composed of ␣ and ␤ subunits that form pentameric ligand-gated cation channels. Nine ␣ subunits (␣2-␣10) and three ␤ subunits (␤2-␤ 4) have been identified in vertebrate neuronal tissues, and different combinatio...