ABSTRACT3 H]epibatidine were found: one that was increased about 4-fold in cells grown for 2 to 4 days in the presence of nicotine and one that was increased 5-fold in cells grown for 2 to 4 days in the presence of nerve growth factor (NGF). The actions of the two treatments were superadditive, resulting in approximately a 13-fold increase in binding sites in cells grown in the combination of the two treatments. The pharmacology of the binding sites in the nicotine-and NGF-treated cells was compared with the pharmacology of defined ␣3␤2 and ␣3␤4 nicotinic acetylcholine receptor (nAChR) subtypes heterologously expressed in human embryonic kidney 293 cells. Nicotine treatment predominantly increased a receptor with characteristics of an ␣3␤2 subtype, whereas the NGF treatment exclusively increased a receptor with characteristics of an ␣3␤4 subtype. Nicotinic receptormediated function measured with the 86 Rb ϩ efflux assay was evident only in the NGF-treated cells, and it had a pharmacological profile that was, again, nearly identical to that of the heterologously expressed ␣3␤4 receptor subtype. Receptor function measured with the [ 3 H]norepinephrine release assay was measurable in both nicotine-treated and NGF-treated cells; however, cytisine-stimulated [ 3 H]norepinephrine release indicated that nicotine treatment increased an nAChR containing ␤2 subunits, whereas NGF increased a receptor containing ␤4 subunits. NGF treatment increased mRNA only for ␤4 subunits in these cells, whereas nicotine treatment did not affect mRNA for any of the subunits measured. After withdrawal of the treatments, the receptors increased by nicotine were much less stable than those increased by NGF.Neuronal nicotinic acetylcholine receptors (nAChRs) are found throughout the central nervous system as well as in autonomic ganglia and the adrenal gland, where they mediate cholinergic neurotransmission critical to the functions of the autonomic nervous system. These receptors are composed of two types of subunits, ␣ and ␤. To date, nine neuronal ␣ subunit genes (␣2-␣10) and three ␤ subunit genes (␤2-␤4) have been found in vertebrate tissues. Different combinations of subunits compose subtypes of nicotinic receptors, all of which are ligand-gated cation channels that conduct Na ϩ , K ϩ , and Ca 2ϩ when activated by the endogenous neurotransmitter acetylcholine or by nicotine or other nicotinic agonists. However, the different receptor subtypes have distinguishing biophysical and/or pharmacological properties, including channel conductances, rates of desensitization and recovery, and sensitivity to drugs (for reviews, see Sargent, 1993;Colquhoun and Patrick, 1997;Lukas, 1998).Certain subtypes of nAChRs are strongly regulated by nicotine. Previous studies have shown that chronic administration of nicotine increases nicotinic receptor binding sites in rat and mouse brain (Schwartz and Kellar, 1983;Marks et al., 1983; for review, see Gentry and Lukas, 2002), and a similar increase is found in human brains from people who smoked tobacco (Benwe...