Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor.

Neuronal nicotinic receptors in PC12 cells were measured by binding with [3H]epibatidine and in functional studies with agonist-stimulated 86Rb+ efflux and [3H]norepinephrine release assays. Two subtypes of receptors labeled by [3H]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 alpha3beta2 and alpha3beta4 nicotinic acetylcholine receptor (nAChR) subtypes heterologously expressed in human embryonic kidney 293 cells. Nicotine treatment predominantly increased a receptor with characteristics of an alpha3beta2 subtype, whereas the NGF treatment exclusively increased a receptor with characteristics of an alpha3beta4 subtype. Nicotinic receptor-mediated function measured with the 86Rb+ 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 alpha3beta4 receptor subtype. Receptor function measured with the [3H]norepinephrine release assay was measurable in both nicotine-treated and NGF-treated cells; however, cytisine-stimulated [3H]norepinephrine release indicated that nicotine treatment increased an nAChR containing beta2 subunits, whereas NGF increased a receptor containing beta4 subunits. NGF treatment increased mRNA only for beta4 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.